Nanoparticulate probe for in vivo monitoring of tissue oxygenation

ABSTRACT

A new class of micro- and nano-particulate paramagnetic spin probes especially useful for magnetic resonance imaging techniques, including electron paramagnetic resonance (EPR) and magnetic resonance imaging (MRI). The probes are lithium phthalocyanine derivative compounds. Also provided are suspensions and emulsions comprising lithium phthalocyanine derivative probes. Also provided are noninvasive methods for measuring noninvasive methods of measuring oxygen concentration, oxygen partial pressure, oxygen metabolism, and nitric oxide concentration in a specific tissue, organ, or cell in vivo or in vitro.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending application Ser. No.12/688,767, filed Jan. 15, 2010, which is a continuation of applicationSer. No. 10/935,297, filed Sep. 7, 2004, now U.S. Pat. No. 7,662,362,which claims the benefit of U.S. Provisional Application No. 60/500,714,filed Sep. 5, 2003, the disclosure of each of which is expresslyincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was funded at least in part by the National Institutes ofHealth, grant CA78886. The government may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION Technical Field

Over the last decade, it has become clear that cigarette smoking induceslung cancer and vascular disease. It is a major risk factor in theoccurrence of heart attack and stroke. Vascular disease leads to tissuedamage including heart attack and stroke and is by far the leading causeof morbidity and mortality in the United States. Tobacco use leads totissue injury in the lungs, heart and vasculature and is implicated inapproximately 20% of all deaths in the United States. Tobacco inducedperipheral vascular disease results in a broad range of medicalcomplications including vascular insufficiency, claudication, stasisulcers, wound formation, impaired wound healing and chronic wounds.

Cigarette smoke has a very high content of free radicals, molecules withunpaired electron spin, that are highly reactive and once present incells and tissues induce lipid, protein and DNA damage. These freeradicals as well as secondary oxygen and nitrogen centered radicals arethe key radical species that trigger tobacco-induced carcinogenesis, aswell as cardiovascular and lung injury. Oxygen radicals can trigger aninflammatory response through leukocyte chemotaxis and activation thatin turn results in a vicious cycle of further oxidant formation andinflammation. Investigators of this program have demonstrated thatoxygen radicals induce cellular proliferation, a key process in thepathogenesis of cancer and atherosclerosis [5].

In just over two decades the advent of magnetic resonance imaging (MRI)has revolutionized the practice of medicine. At an ever-acceleratingrate MRI has achieved breakthroughs first in enabling high-resolutionanatomical imaging of tissue abnormalities in disease and more recentlyalterations in organ function. With the advent of molecular medicine andtargeted therapeutics as well as the breakthroughs in the sequencing ofthe human genome, it has been realized that potentially the next evenmore powerful horizon for magnetic resonance imaging is in the imagingof molecular and gene expression that will enable the early detection orprevention of disease as well as facilitate the treatment and cure ofexisting illness.

Electron paramagnetic resonance (EPR) has advantages over proton NMR inthat it is inherently over 1,000 times more sensitive on a spin basisand furthermore, for a given frequency, measurements may be performed atmuch lower magnetic fields enabling the use of low-cost magnet systems.Over the last several years, it has been shown that the electronspin-based technique of EPR imaging (EPRI) can provide high sensitivityand high resolution images of paramagnetic materials. For example at1200 MHz it was shown that concentrations as low as 10 nM could bedetected for a typical nitroxide spin label and this sensitivity is atleast two orders of magnitude above that achievable even with ultrahigh-field proton MRI [1]. In addition, it was shown thathigh-resolution 3D images may be obtained with submillimeter resolution.In addition to direct EPR detection of paramagnetic spin probes, thehybrid EPR/NMR technique of Proton Electron Double Resonance Imaging(PEDRI) can also detect paramagnetic probes by the marked Overhauserenhancement observed in proton MRI signal seen upon irradiation of theelectron spin. Enhancements of over 100 fold may be achieved. Theseenhancements translate into markedly improved image quality, contrastand resolution in biological tissues. With this marked enhancement,proton magnetization and image quality even at relatively low fields canexceed that of the highest field MRI systems. For example, in principle,PEDRI image quality at 0.2 T could exceed that at 20 T, if indeed suchan ultra high-field system could be built.

With recent technological advances, it has become possible to imagethese critical free radical mediators of disease using novel magneticresonance imaging techniques. Advances in the magnetic resonance imagingtechniques of in vivo Electron Paramagnetic Resonance Imaging (EPRI) andProton Electron Double Resonance Imaging (PEDRI) have enabled theimaging of these critical mediators of disease and the redox stress theycause in living animals and most recently in man [2, 3, 6, 7]. These MRtechniques along with new types of spin probes and spin traps as well asinnovative nanoparticulate probes have enabled the imaging of freeradicals, oxygen and nitric oxide [1, 8-13]. These breakthroughs havethe potential to revolutionize the diagnosis and treatment of humandisease. Beyond their diagnostic power, spin traps have great potentialfor the treatment of disease since they can trap or scavenge freeradicals preventing radical-induced molecular and cellular damage. Freeradicals, both extrinsic as from cigarette smoke, or intrinsic, frominflammatory stress, are central in the pathogenesis of human diseaseincluding: heart attack, stroke, cancer, neurodegenerative diseases,emphysema/obstructive pulmonary disease as well as the process of aging.The ability to trap and scavenge these critical mediators of disease hasthe potential to revolutionize current medical diagnosis and treatmentand provide the long-awaited cures to a variety of the diseases thathave plagued mankind.

While a great wealth of information may be obtained from the imaging ofintrinsic protons, to achieve MR-based imaging of molecular and geneexpression, there is a critical need for new imaging agents that may bedesigned or targeted to visualize specific molecular targets. There isalso a need for probes that can be tagged to proteins or DNA, enablinggeneralized biomolecular and gene imaging. There is further a need, inaddition to detecting these materials through their effects on protonrelaxation, for the ability to directly detect paramagnetic materialsusing the MR technique of Electron Paramagnetic Resonance (EPR) or otherMR techniques. Additionally, there is a need for new particulate probesthat may be used to accurately determine oxygen concentration in cells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a new class of particulate probes thatare especially useful for magnetic resonance imaging techniques. Theparticulate probes are nanoparticulate and microparticulate probescomprising paramagnetic spin probes that are especially suitable for usewith magnetic resonance (MR) techniques, particularly, but not limitedto, electron paramagnetic resonance (EPR) and magnetic resonance imaging(MRI). The nanoparticulate and microparticulate probes comprise radicalsof lithium phthalocyanine derivative compounds, which include lithiumphthalocyanine derivatives, lithium naphthalocyanine derivatives, andlithium anthraphthalocyanine derivatives.

The probes preferably have a size of 10 microns or less, more preferablyfrom 0.22 to 10 microns, and for intravenous applications, even morepreferably less than 0.22 microns. The probes may be used with a varietyof MR spectroscopy and MR imaging techniques, including but not limitedto magnetic resonance imaging (MRI); electron spin resonance (ESR);electron paramagnetic resonance (EPR); electron paramagnetic resonanceimaging (EPRI); and proton electron double resonance imaging (PEDRI).

The probes of the present invention comprise ligands, dilithiumcomplexes, and lithium radicals. Some preferred dilithium complexes areshown as compounds 1-6:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)_(n)CH₃, O(CH₂)_(n)CH₂OH, O(CH₂)_(n)CH₂NH₂, O(CH₂)_(n)CH₂SH, andcombinations thereof; wherein n is 1-6. Preferred lithium radicals areobtained from these dilithium complexes by electrochemical or chemicaloxidation.

Also provided are suspensions and emulsions comprising lithiumphthalocyanine derivative radicals, which have an oxygen center, makingthem useful for various in vivo and in vitro measurements. Thesuspensions of the present invention are in a media selectednonphysiological media, physiological media, buffers, and combinationsthereof. The particulate probes are selected from the group consistingof:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)_(n)CH₃, O(CH₂)_(n)CH₂OH, O(CH₂)_(n)CH₂NH₂, O(CH₂)_(n)CH₂SH, andcombinations thereof; and wherein n is 1-6; and combinations thereof.

The suspensions of the present invention further comprise a stabilizingagent and/or a stabilizing media. Some preferred stabilizing agents areselected from, but not limited to amino acids, synthetic peptides,peptides of natural origin, proteins, sugars, carbohydrates, nucleicacid homopolymers, amino acid homopolymers, DNA, RNA, other bipolymers,and combinations thereof. The stabilizing agents adhere to the radicalprobe without blocking the oxygen active centers. Some preferredstabilizing media include, but are not limited to emulsions containingsaturated fatty acids; emulsions containing unsaturated fatty acids;emulsions containing saturated and unsaturated fatty acids; salts ofemulsions containing saturated fatty acids; salts of emulsionscontaining unsaturated fatty acids; salts of emulsions containingsaturated and unsaturated fatty acids; diglycerides; triglycerides; bilesalts; and combinations thereof.

The suspensions of the present invention may further containphospholipid, wherein the phospholipid encapsulates the radical probewithout blocking the oxygen active centers. The phosholipid may formphospholipid liposomes which encapsulate the radical probe withoutblocking the oxygen active centers. Some preferred phospholipidsinclude, but are not limited to cholesterol, phosphatidyl choline,phosphatidylethanolamine, phosphatidylserine, cardiolipin, andcombinations thereof; and wherein the phospholipid is in the form ofunilamellar or multilamellar liposomes or vesicles.

Further provided are noninvasive methods of measuring oxygenconcentration, oxygen partial pressure, or oxygen metabolism in aspecific tissue or organ in a subject, the method comprising the stepsof: (a) administering a lithium phthalocyanine derivative radical probeto the subject; and (b) applying a magnetic resonance (MR) spectroscopytechnique capable of measuring O₂ concentration in tissues or organs ofthe subject. Additionally, the probes of the present invention may beused to measure nitric oxide (NO) concentration, separate from or alongwith oxygen concentration, using the same method.

Preferred lithium phthalocyanine derivative radical probes include, butare not limited to:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)_(n)CH₃, O(CH₂)_(n)CH₂OH, O(CH₂)_(n)CH₂NH₂, O(CH₂)nCH₂SH, andcombinations thereof; wherein n is 1-6; and combinations thereof.

The lithium phthalocyanine derivative radical probes are useful for MRspectroscopy and MR imaging, particularly, but not limited to MRI, ESR,EPR, ERPI, and PEDRI. The probes may be delivered to a subjectintravenously or may be implanted into tissue. The probes are useful forstudying tissues, organs or cells. When the radical probe is deliveredto the subject intravenously, it may be delivered as a suspension oremulsion. The probe may also be delivered directly to the tissue ororgan of interest. When injected into the tissue of interest, theradical probes may remain active in a subject for up to 12 months, andpreferably remain active for more than 180 days, allowing study of thesame tissue or organ over an extended period of time.

The radical probes may be attached to a peptide or glycoconjugate thathas specific affinity for cell surface markers, wherein the radicalprobe acts as a cell migration marker. The radical probes may also to anantibody, wherein the antibody has an affinity to cell surface proteinsthat lead as markers of cell migration, cell division, and cell death.The radical probes may also be internalized in live cells, either invivo or in vitro for the study of intracellular oxygenation, cellularhypoxia, cellular hyperoxia, cell division, cellular migration, ormetastatis. The radical probes may also be utilized to study thekinetics of enzymes that involve oxygen consumption and release inorgans, tissues, or cells, in vivo or in vitro. The subjects may be anysubject of interest. Preferably, the subject is a human subject. Themethods of the present invention may also be used to study microbialoxygen metabolism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Molecular structure of lithium octa-n-butoxynaphthalocyanine(LiNc-BuO) radical. The neutral radical is paramagnetic and prepared asa microcrystalline solid.

FIG. 2: EPR spectrum of LiNc-BuO nanocrystalline powder suspended inPBS. The spectrum (A) was measured at X-band (9.78 GHz) from a 10 μL ofthe suspension equilibrated with 10% (pO₂: 76 mmHg) oxygen at roomtemperature. The instrumental settings were: microwave power, 1 mW;modulation amplitude, 63 mG; modulation frequency, 100 kHz; receivertime constant, 82 msec; acquisition time, 60 sec (4×15 sec scans); Asingle sharp peak is observed with peak-to-peak width (Ab_(pp)) of 852mG. Also superimposed on this spectrum is a computer fit that wascalculated assuming Lorentzian line-shape. The difference between themeasured spectrum and the Lorentzian fit is shown in (B) at 4×magnification. The difference curve shows only noise suggesting that theline-shape is 100% Lorentzian (R2=0.9999).

FIG. 3: Effect or oxygen concentration (pO₂) on the peak-to-peak EPRline-width (Ab_(pp)) of LiNc-BuO particulates. The particulates weresuspended in PBS equilibrated with mixtures of oxygen/nitrogen gases.The spectra were acquired as described in FIG. 2. The line-widthincreases linearly with pO₂ in the range 0 to 760 mmHg (corresponding to0-100% oxygen at I atmospheric pressure) with an anoxic at 0% oxygen)line-width of 210 mG and slope (oxygen sensitivity) of 850 mG/mmHg. Theeffect or oxygen on the line-width was highly reversible andreproducible under a variety of conditions.

FIG. 4: Long-term stability and response to oxygen, in vivo. Thestability of LiNc-BuO particulates implanted in the gastrocnemius muscle(upper hind leg) of C3H mice was studied up to 180 days. The plot showsrepeated measurements of pO₂ from a single mouse. The response ofparticulates to oxygen was checked by temporarily constrictingblood-flow to the leg. The data shows that the particulates are stableand responsive in the live tissues up to 6 months. The spectra shownabove were from a mouse on day 180 after implantation of theparticulate. The spectra were acquired as described in FIG. 2.

FIG. 5: In vivo measurements of pO₂ from tumor and normal gastrocnemiusmuscle tissues in mice as a function of tumor growth in mice with RIF-1tumor. LiNc-BuO particulates were unplanned in the tumor (RIF-1) onright leg and normal muscle on left leg and the tissue. pO₂ values wererepeatedly measured on the same animals up to 8 days after implantationof the particulates. Mean values of pO₂ (A) and tumor volume (B)recorded repetitively from 7 mice are shown. The tumor pO₂ decreasedcontinuously to ˜2 mmHg on day 8 after implantation, while the normalmuscle pO₂ remained almost constant (17.6±25 mmHg) during the sameperiod.

FIG. 6: Internalization of the LiNc-BuO particulates in cells. Theparticulates (<2 μm) were coincubated with the human arterial smoothmuscle cells for 72 h followed by repeated washings as described in theDetailed Description of the Invention. The cells were photographed underan inverted microscope while still adherent to the substratum of the 35mm dish. The LiNc-BuO particulates are seen as dark green crystalsinside cells.

FIG. 7: EPR spectra of LiNc-BuO microcrystalline powder suspended insaline at various partial pressures of molecular oxygen. The spectrawere measured at X-band (9.78 GHIz) from a 20 μL of the suspensionequilibrated with 0% (pO₂ mmHg) and 20.9% (pO₂: 159 mmHg) oxygen at roomtemperature. The instrumental settings were: microwave power, 1 mW,modulation amplitude, 63 mG, modulation frequency 100 kHz, receiver timeconstant 82 msec; acquisition time 60 sec (4×15 sec scans). A singlesharp peak is observed with a peak-to-peak width (ΔB_(pp)) of 210 mG at0% oxygen and 1550 mG at 20.9% oxygen.

FIG. 8: Time dependence of pO₂ in mouse aortic endothelial cellsuspensions exposed to various agents and treatments. The pO₂measurements were performed on 20,000 cells taken in a 20 μL capillarytube (id: ×μm; cell density: 1×10⁶ cells mL) by EPR spectroscopyutilizing LiNc-BuO oximetry probe. The capillary tube was sealed at bothends and pO₂ measurements were performed continuously for up to 20 min.(a) Control (b) KCN (100 μM), (c) rotenone (100 μM), (d) DPI (100 μM),(e) menadione (50 μM), (f) LPS (10 μg/ml). Values at each time point areexpressed as mean±SD of 4-5 independent experiments. The solid linesthrough each data set show the linear variation of pO₂, which suggestsconstancy in oxygen consumption as a function of time.

FIG. 9: Effect of menadione on the rate of oxygen consumption by mouseaortic endothelial cells. The measurements were performed as describedin the Detailed Description and oxygen consumption rates are calculatedfrom the slope of change of pO₂ with time. Cells were treated with 10,50, 100 and 200 μM concentration of menadione and the measurements werestarted immediately after adding menadione. Values are ±SD of 5experiments. *p<0.001 versus control; **p<0.001 versus control.

FIG. 10: Effect of lipopolysaccharide (LPS) on the rate of oxygenconsumption by MAECs. The measurements were performed as in FIG. 2 andoxygen consumption rates were calculated from the slope of change of pO₂with time. Cells (1×10⁶ Cells/ml) were treated with 10 or 20 μg/mlconcentration of LPS and measured either immediately after mixing orafter 2 h or incubation of the mixture under aerobic conditions. Valuesare mean±SD of 5 experiments. *p<0.05 versus control.

FIG. 11: Oxygen consumption rates in mouse aortic endothelial cellsuspensions exposed to various agents and treatments. Cells (1×10⁶Cells/ml) were treated with menadione (50 μM), LPS (10 μg/ml), KCN (100μM), rotenone (100 μM) and DPI treatment. Values are mean±SD of 5experiments. *p<0.001 versus control; **p<0.01 versus control.

FIG. 12: EPR spectrum of a suspension of LiNc-BuO and TAM (10 μM) in PBS(pH 7.4) equilibrated with room air (20.9% oxygen). The originalspectrum (top) is a composite of two components: a sharp peak from TAM(g+2.0030) and a broad peak from KiNc-BuO (g+2.0024). The additionalpeaks indicated by * on both sides of the spectrum are due to 13 Chyperfine from TAM (35). EPR data acquisition parameters were:modulation amplitude, 100 mG; microwave power 1 mW; time constant,8-msec, scan time, 15 s. The computer fit (middle) shows thedecomposition of the original spectrum into two components, that theLiNc-BuO and TAM. The computer fit (sum of the two components) issuperimposed onto the original spectrum. The residual (bottom) curveshows the difference between the original and computer fit (R²=0.9977).

FIG. 13: Photomicrograph of bovine lung microvascular endothelial cellsshowing internalization of the LiNc-BuO microparticulates. The LiNc-BuOparticulates are seen as dark green crystals inside the cells.

FIG. 14: Effect of oxygen concentration (pO₂) on the peak-to-peak EPRline-width (ΔBpp) of LiNc-BuO and TAM. Measurements were madeindependently of LiNc-BuO microcrystalline particulates suspended insaline and TAM (10 μM) in PBS equilibrated with mixtures ofoxygen/nitrogen gases. The spectra were acquired as describe din FIG. 1.The line-width increases linearity of pO₂ in the range of 0 to 160 mmHg)with an anoxic (0% oxygen) line-width of 210 mG and slope (sensitivity)of 8.5 mG/mmHg for LiNc-BuO and an anoxic line-width of 148 mG and slope(sensitivity) of 0.36 mG/mmHg for TAM.

FIG. 15: Extracellular and intracellular measurement of pO₂ in bovinelunch microvascular endothelial cells (BLMVECs). Intracellular pO₂ wasmeasured using internalized LiNC-BuO particulated in BLMECs. Theextracellular pO₂ was measured simultaneously using 10 μM TAM.Measurements were made at room air (20.9% or pO₂: 159 mmHG) and at 7.5%(pO₂: 57 mmHG) oxygen. Values are mean±SD of 5 experiments. *p<0.001versus extracellular pO₂.

FIG. 16: Effect of menadione and cyanide on intracellular andextracellular pO₂ in BLMVECs. The pO₂ measurements were made in cellstreated with menadione (50 μM) and potassium cyanide (100 μM). Themeasurements were performed as in FIG. 4. Values are mean±SD of 5experiments.

FIG. 17: Simplified molecular structure of Li(OBu)₈Nc used for the DASHanalysis. Eighty eight hydrogens and one lithium were removed from theoriginal molecule. The side lengths of the naphthalocyanine rings are17.9˜18.0 Å in NiNc, CuNc, and ZnNc.

FIG. 18: Positional parameters obtained from initial DASH trial runswhere no constraints were used. Black open circles are from trials usingLi(OBu)₈Nc (20 results with lowest χ_(pro) ², out of 45 trials) and opentriangles are form the trials using Li(OBu)₈Nc (reproducibility, 10/10),Li(OMe)₈Nc (8/10), Li(OEt)₈Nc (9/10), and Li(OPr)₈Nc (6/10). For thegroups marked as I and II, average and deviations of the coordinates are(0.051±0.021, 0.026±0.019, 0.368±0.031) and (0.443±0.019, 0.024±0.023,0.151±0.023), respectively.

FIG. 19: Simulated annealing refinement profile of the XRPD patter forLi(OBu)₈Nc. Calculated (solid line) and observed (cross) data areoverlapped. Bragg reflection positions and the difference pattern areshown below.

FIG. 20: Stacking patter of Li(OBu)₈Nc as determined from the DASHanalysis, viewed along (a) a-, (b) b-, and (c) c-axes. The rectanglesrepresent the cross sections of infinite channels propagating in theviewing direction (their sizes are mentioned in text).

FIG. 21: (Top) LeBail and (bottom) Pawley fits to the XRPD pattern ofLi(BuO)₈Nc. Observed (cross) and calculated data (solid line) areoverlapped, and the difference pattern and expected peak positions areshown. In the LeBail fit, background is also shown.

FIG. 22: Simulated annealing refinement profile of the XRPD pattern forLi(OBu)₈Nc, between 3.2-32°. Calculated (solid line) and observed(cross) data are overlapped. Bragg reflection positions and thedifference pattern are shown below.

DETAILED DESCRIPTION OF THE INVENTION

NMR-based magnetic resonance imaging, MRI, enables visualization of thedistribution of nuclear spins, mostly protons, in tissues. It has becomea ‘gold standard’ for noninvasive diagnosis of tissue abnormalities.Electron paramagnetic resonance imaging (EPRI) is a parallel technology,which enables visualization of the distribution of electron spins (freeradicals) in tissues. EPR is inherently about 3 orders of magnitude moresensitive than NMR. It can directly detect and image relatively stablefree radicals as well as labile radicals such as oxygen-derivedsuperoxide and hydroxyl free radicals that are implicated in thepathogenesis of oxidant injury [14-21]. Recently, EPR methods have alsobeen developed to enable detection of nitric oxide [22-24]. In addition,spin probes may be used to image cellular radical metabolism and redoxstate, membrane structure and fluidity, oxygen, pH, temperature, proteinstructure, and cell death. With spin labeling of molecules and cells,noninvasive mapping of their localization in tissues may be performed[8, 9, 12, 25-34]. Recent advances in magnetic resonance instrumentationand probe design have enabled integration of these two modalities into anew technology, proton electron double resonance imaging (PEDRI) whichis capable of co-imaging free radicals and protons [4, 7, 35].

A major power of EPR technology is its ability to precisely measure O₂in tissues [6, 9, 31, 36-40]. This ‘EPR oximetry’ technique uses spinprobes whose EPR line-widths are highly sensitive to O₂ concentration.It enables precise and accurate measurements of O₂ concentrations intissues, noninvasively and repeatedly over periods of weeks from thesame site. This approach uses fine crystals (nanoprobes) ofphthalocyanine-based radical molecules that are stacked producing a verystrongly exchanged-narrowed EPR line-shape, that is highly sensitive tolocal O₂ concentration [9, 40, 41]. These nanoprobes are biocompatibleand stable in tissues. They may be implanted at the desired site or witha suitable coating may be infused into the vasculature for targeteddelivery to tissues. In addition, we recently demonstrated that thesenanoprobes may be internalized in cells enabling measurement ofintracellular pO₂ with milliTorr accuracy.

Cellular redox measurements are performed using redox-sensitive nitroxylmolecules that are soluble spin probes [36]. A variety of nitroxylmolecules capable of reporting cellular redox levels including totalredox, thiols, and glutathione may be used. These molecules are nontoxicand are converted to nonradical species and cleared from the systemwithin hours after infusion.

Overall, the novel in vivo MR techniques of EPRI and PEDRI can provideimportant information about tissue radical generation, oxygenation,nitric oxide production, metabolism and injury as well as therapeuticdelivery. With the recognized importance of free radicals, oxygen and NOin disease this information is of crucial importance. These techniquesalso can enable high sensitivity measurement of molecular expression,gene expression, cell therapy and the delivery of a broad range ofmolecular therapeutics. These major advances in molecular and geneticimaging have the potential to revolutionize medical diagnosis andtreatment.

ABBREVIATIONS

The following abbreviations are used herein:AAPH—2,2′-azobis(2-amindinopropane)dihydrochloride; CRISP—Crystallineinternal spin probe; DMEM—Dulbecco's modified Eagle medium; EPR—Electronparamagnetic resonance; FBS—Fetal bovine serum; HASMC—Human arterialsmooth muscle cells; LiNc—Lithium naphthalocyanine; LiNc-BuO—Lithium5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine; LiPc—Lithiumphthalocyanine; MEM—Minimal essential medium;Nc-BuO—5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine;PBS—Phosphate-buffered saline; pO₂—Partial pressure of oxygen;RIF-1—Radiation-induced fibrosarcoma-I;SNAP—S-nitroso-N-acetyl-penicillamine; and TAM—Triarylmethyl.

The probes of the present invention are lithium phthalocyaninederivatives. As used throughout the specification and claims, “Lithiumphthalocyanine derivatives” includes, but is not limited to lithiumphthalocyanine derivatives and radicals thereof; lithiumnaphthalocyanine derivatives and radicals thereof; and lithiumanthraphthalocyanine derivatives and radicals thereof. The probes of thepresent invention are designed or targeted to visualize specificmolecular targets. These probes may also be tagged to proteins or DNAenabling generalized biomolecular and gene imaging. The probes may beimplanted at a desired site or coated with a suitable coatingformulation may be infused into the vasculature for targeted delivery totissues to facilitate study of a tissue of interest. The probes of thepresent invention may also encapsulated in phospholipid liposomes (e.g.phosphatidylcholine and cholesterol) to facilitate rapid uptake into thecells, which are delivered into cells engineered for tissue or woundrepair.

The lithium phthalocyanine derivatives are particulate, and have lowsolubility in aqueous solutions as well as in common organic solvents,making them particularly suitable for the following applications: (i) asan oxygen-sensing EPR probe for accurate determination of concentrationof oxygen and (ii) as a molecular and cellular imaging probe for EPR/MRImethods. The probes also have applications in the field of biomedicalresearch and clinical studies, including, but not limited to: (1)determination of oxygen concentration in tissues; (2) determination ofoxygen concentration in cells; (3) determination of oxygen consumptionby cells; (4) targeted intracellular delivery of particulate oximetryprobes; and (5) DNA or protein-targeted spin probes. Additionalapplications of the nanoparticulate probes of the present inventioninclude cell-tagging and cell-tracking applications; studying cancermetastasis in experimental models; tissue engineering (stem cellresearch); tagging antibody; MRI contrast agent; implantableoxygen-sensor in peripheral vascular disease; oxygen-sensor in woundhealing applications; and implantable oxygen-sensor in cancer therapy.

Synthesis of Micro and Nanoparticulate Oximetry Spin Probes Based onPhthalocyanine Macrocycles:

Mono-lithiated phthalocyanine and naphthalocyanine derivatives aresynthesized using chemical or electrochemical procedures as we reportedpreviously [11, 42]. The synthesis is set forth in the followingsynthetic schemes:

The spin probes are prepared as microcrystalline particles andcharacterized using X-ray diffraction and magnetic susceptibilitytechniques. The particles are suspended in complete medium containing10% serum and sonicated with a probe sonicator. Alternatively theparticles may be sonicated in presence of 1 mg/mldioleoylphosphatidylcholine or lecithin to entrap the particles inliposomes and filtered through 0.22-10 micron filters to separate themaccording to the size. The EPR properties including oxygen sensitivityof the suspension are verified for each batch.

Several approaches known to those skilled in the art are used to furtherdevelop stable nanoprobe suspensions for iv or other systemic use. Thefollowing are taken into consideration in the development:biocompatibility (non-toxicity), preservation of oxygen sensitivity, andlong-term shelf- and tissue stability in solutions of high ionicstrength. In general, the suspension formulation requires a stabilizingagent, which adheres to the surface of the particle probe withoutblocking the oxygen active centers, which are responsible for the oxygenadsorption. The conformation and surface distribution of the agent oradditive that is used to stabilize the suspension is taken intoconsideration. Fabrication of fine suspensions then requires milling ofthe probe particle using a ball mill and a homogenizer. Conventionalmethods of preparation of water-based colloidal dispersions usingwater-soluble stabilizers (low molecular weight surfactants like heparinand Tween; water-soluble polymers like pluriol), similar to described byGallez et al. [43, 44], may be used. Alternatively, dispersions may beprepared using organic solvent-based systems with water-insolublestabilizers, and subsequently transforming them to aqueous system byadding water and removing the organic solvent. Another approach is tosynthesize submicrometer-sized silica oxide suspension (silica gel) inthe presence of the probe particles. This can produce coaggregates ofthe probe and silica dioxide. Smaller particles of silica gel can forman oxygen-permeable shell and thus protect the probe particles fromforming bigger aggregates. Submicron <0.22 micron) particulates withbiocompatible coating formulations will be developed for iv infusion.

Targeted Intracellular Delivery of Particulate Oximetry Probes:

Specially synthesized particulate oximetry nanoprobes, encapsulated inphospholipid liposomes (phosphatidylcholine and cholesterol) tofacilitate rapid uptake into the cells, may be delivered into cellsengineered for tissue or wound repair. Ligand-targeted liposomes andlipoplexes are highly useful as cargoes to deliver the nanoprobes todesignated cell types in vivo [45-51]. Different types of ligands, suchas receptors, peptides, vitamins, oligonucleotides or carbohydrates maybe positioned onto the liposomal surface which will enhance the bindingaffinity [52-55]. The nanoprobe containing cells are characterized usingoptical confocal microscopy and EPR spectroscopy. Internalizationtechniques may be developed for the different stem or other cells topromote wound repair and used to image intracellular oxygenconcentration, cell migration and proliferation of the targeted celltherapy.

DNA or Protein-Targeted Spin Probes:

As per the approach of Kursa et al. [56], novel shieldedtransferrin-polyethylene glycol-polyethylenimine DNA or proteincomplexes ligated with EPR probes (redox, NO, pH and O₂ probes) may bedeveloped and used to study gene or peptide/protein delivery andconcomitant localized oxygen and radical metabolic events. The use andability of nonviral DNA complexes to deliver genes to cells and tissuesin vivo offers a potential for delivery of DNA specific EPR probes [51,57, 58]. Cell replication and cell cycle-specific gene expression inconcert with oxygen and radical metabolism may be studied by deliveringcomplexes that will be generated by mixing plasmid DNA, linearpolyethylenimine, PEG and transferrin that provides a ligand forreceptor-mediated cell uptake [57]. Cells in culture or in situ may beloaded with these complexes with a specific therapeutic gene (DNA) ofinterest and the oxidative and redox metabolism mapped.

Toxicology and Pharmacokinetic Evaluations as Required for IND and FDAApproval:

Many of the paramagnetic molecules under development in this programhave been extensively tested in animals and some even in human studies.Even at very-high applied concentrations of up to 150 mM no toxicity hasbeen seen. With carbon-based micro and nanoparticulates there is ahistory of human application as in the marking of surgical fields withIndia ink. The naphthalocyanine particulates of the present inventionhave been chronically studied in small animals and no adverse effect ortoxicity has been seen acutely or up to 3 months. We recognize the needfor pharmacokinetic and toxicological testing particularly for systemicformulations.

Derivatives of Phthalocyanines:

Phthalocyanine is planar macro cycles that contain four isoindole unitsand present 18 pi electrons cloud delocalized over an arrangement ofalternating carbon and nitrogen atoms. The unique property ofphthalocyanine comes from the electron delocalization, which can beeasily modified by introducing a variety of structural alterations.

In addition to the prototype probe (LiNc-BuO) the present inventionencompasses a series of substituted derivatives (R groups) andbenzo-annulated derivatives (phthalo, naphthalo, and anthraphthalo) asillustrated in structures 1-18. In general, the derivatives of thepresent invention include: (i) Four different R groups: O—(CH2)n-CH₃,where n=1-6; O—(CH₂)_(n)—CH₂OH, where n=1-6; O—(CH₂)_(n)—CH₂NH₂, wheren=1-6; O—(CH₂)_(n)—CH₂SH, where n=1-6; and combinations thereof; (ii)two different attachments/positions: Para and Ortho, and (iii) threedifferent benzo-annulations: phthalocyanine, naphthalocyanine, andanthraphthalocyanine.

The compounds may be synthesized using any appropriate chemical orelectrochemical procedures. The synthetic procedures will be optimizedfor each specific case. Syntheses for compounds 1-18 are shown, andsuitable modifications can easily be made by those skilled in the art.

The Design of Lithium Phthalocyanine Derivatives as Oxygen-Sensors:

Lithium metal has high mobility and smaller size compared to otheralkali metals such as Na or K. This enables the metal ion to site in thecenter of the macro cycle allowing very tight stacking of the moleculesin the crystal. This close packing results in highly exchange-coupledsystem with extremely narrow EPR lineshape. The benzo-annulations extendthe delocalization of the unpaired electron in the molecule, which mayreduce dipole-dipole interactions between the unpaired electrons in thestacked molecules. The absence or minimization of dipole-dipoleinteraction is highly preferred to obtain pure Lorentzian lineshapes.The substituents (R groups) are used as handles (i) to modulate themolecule-molecule distance in the crystal enabling different lineshapesensitivities to oxygen, (ii) to impart hydrophobicity to theparticulate so that the material can be internalized or stabilized inbiological tissues, (iii) to vary the inter-stack bore size in thecrystal enabling desired molecules such as oxygen to freely diffuse intothe crystal lattice and (iv) to establish anchor points to attachspecific molecules to the crystal. Thus the probes may be used for arange of biological applications as discussed below.

The lithium phthalocyanine derivatives, have low solubility in aqueousand common organic solvents. Unsubstituted annulated phthalocyanines arepractically insoluble in organic solvents and are hard to purify andrecrystallize. In order to have their functionality most effective, thelithium phthalocyanine derivatives with increasing solubility in organicsolvents is planned. The insolubility of metal phthalocyaninederivatives results from their molecular stacking, which gives rise tostrong intramolecular interaction between the macrocycles inphthalocyanine molecules. The introduction of long substituents in themacro cycle increases the solubility of the metal phthalocyaninederivatives.

Calculations of the electronic properties of annulated phthalocyaninesshow that linear annulations of benzene rings produces a continuousdestabilization of the HOMO level and narrowing of the HOMO-LUMO energygap. One-dimensional stacks of the linearly annulated phthalocyaninesare calculated to have lower oxidation potentials and narrower gap thanangularly annulated systems. These theoretical results are confirmed bystudies on 1,2-naphthalocyanine, 9,10-phenanthrenocyanine and2,3-naphthalocyanine and the corresponding bridged stacked systems.Benzoannulation with electron-releasing groups such as alkoxy group oflithium phthalocyanine increases the electron delocalization, andincrease in spin electron intensity in the macrocycles.

In accordance with the present invention, the phthalocyanine orannulated phthalocyanine moieties may also be functionalized with groupssuch as hydroxyl, thiol or amino group. This phthalocyanine moiety maythen be tagged with many biologically important molecules for detectionby EPR spectroscopy and imaging.

The particulates are paramagnetic spin probes with very high spindensity. The particulates are especially suitable for the followingapplications: (i) as an oxygen-sensing EPR probe for accuratedetermination of concentration of oxygen and (ii) as a molecular andcellular imaging probe for EPR/MRI methods. The probes may be used formany different applications in the field of biomedical research andclinical studies as set forth in the next several paragraphs.

(1) Determination of Oxygen Concentration in Tissues:

Electron paramagnetic resonance (EPR)-based oxygen measurements(oximetry) coupled with particulate probes have some unique advantagesover the other methods. The particulate EPR probes for oximetry have thefollowing advantages: (i) they report pO₂, which is a better parameterin a heterogeneous cellular system (ii) they do not consume oxygen (iii)they provide higher resolution at lower pO₂ and (iv) they are stable incells and tissues for repeated measurements of oxygen tensions withoutreintroduction of the probe. The probe may be implanted in the desiredlocation of the tissue and repeated measurements of tissue oxygenationmay be performed over a period of several months. The measurements areaccurate, reliable and noninvasive.

(2) Determination of Oxygen Concentration in Cells:

In view of the importance of critical oxygen concentration in cells foroperation of normal cellular events, methods capable of determining theoxygen concentration in cells and tissues are highly crucial. Althoughmany methods are available to measure oxygen concentration in cells,each method has its advantages and disadvantages, and no single methodis completely satisfactory for cellular studies The pththalocyaninemicro/nano crystals can be easily internalized in cells by endocytosis.This will enable determination of oxygen concentration in cells. Thehigh spin density of the particulates can enable measurements in asingle cell.

(3) Determination of Oxygen Consumption by Cells:

Cellular oxygenation and oxygen consumption rate (OCR) are importantphysiological and metabolic indicators of cellular function. Normalcellular function and homeostasis require a critical level of oxygenconcentration (measured as oxygen tension, pO₂) in the cells to providean adequate supply of oxygen for the mitochondrial oxidativephosphorylation process.

We have previously demonstrated that the octa-n-butoxy derivative ofnaphthalocyanine neutral radical (LiNc-BuO) enables accurate, preciseand reproducible measurements of pO₂ in cellular suspensions. In thecurrent study, we carried out measurements to provide an accuratedetermination of pO₂ in small volume with less number of cells (20,000cells) that has not been possible with other techniques. This studyclearly demonstrated the utilization of EPR spectrometry with LiNc-BuOprobe for determination of oxygen concentration in cultured cells.

Additional applications include, but are not limited to: cell-taggingand cell-tracking applications; studying cancer metastasis inexperimental models; tissue engineering (stem cell research); taggingantibody; MRI contrast agent; implantable oxygen-sensor in peripheralvascular disease; oxygen-sensor in wound healing applications; andimplantable oxygen-sensor in cancer therapy.

NMR-based magnetic resonance imaging, MRI, enables visualization of thedistribution of nuclear spins, mostly protons, in tissues. It has becomea ‘gold standard’ for noninvasive diagnosis of tissue abnormalities.Electron paramagnetic resonance imaging (EPRI) is a parallel technology,which enables visualization of the distribution of electron spins (freeradicals) in tissues. EPR is inherently about 3 orders of magnitude moresensitive than NMR. It can directly detect and image relatively stablefree radicals as well as labile radicals such as oxygen-derivedsuperoxide and hydroxyl free radicals that are implicated in thepathogenesis of oxidant injury. With spin labelling of molecules andcells, noninvasive mapping of their localization in tissues may beperformed [59-62].

A major power of EPR technology is its ability to precisely measuremolecular oxygen in tissues [61]. This ‘EPR oximetry’ technique usesspin probes whose EPR line-widths are highly sensitive to O₂concentration. It enables precise and accurate measurements of O₂ intissues, noninvasively and repeatedly over periods of weeks from thesame site. The approach uses fine crystals (nano/microparticulates) ofphthalocyanine-based radical molecules that are stacked to produce avery strongly exchanged-narrowed EPR line-shape that is highly sensitiveto local O₂ concentration. The EPR line-shape of these nanoprobes ishighly O₂ sensitive, and they are biocompatible and stable in tissues.They may be implanted at the desired site or with a suitable coatingformulation can be infused into the vasculature for targeted delivery totissues. In addition, we recently demonstrated that these nanoprobes canbe internalized in cells enabling measurement of intracellular pO₂ withmilliTorr accuracy

The newly developed nanoparticulate EPR imaging technology is especiallyuseful (i) to visualize and track the migration of endothelialprogenitor cells and (ii) to simultaneously measure intracellularoxygenation. This is done by internalizing the paramagneticnanoparticles (size <200 nm) by derivatizing with Tat protein-derivedpeptide sequences as reported by Lewin et al. [63]. The internalizedcells may be characterized using optical confocal microscopy and EPRspectroscopy. The lithium naphthalocyanine (LiNc-BuO) spin probe, whichhas very high EPR spin density and is readily internalized in cells, isused. Imaging of the distribution of particles is performed in vivousing low-frequency (1.2 GHz) EPR imager. Measurement and mapping ofintracellular oxygen concentration will be performed as we reportedpreviously [64].

Additionally, the inventive particulates are suitable for the followingapplications for targeted intracellular delivery of particulate oximetryprobes, and as DNA or protein-targeted spin probes, as discussed above.

Novel Particulate Spin Probe for Targeted Determination of Oxygen inCells and Tissues:

The synthesis and characterization of a new lithiumocta-n-butoxy-substituted naphthalocyanine radical probe (LiNc-BuO) andits use in the determination of concentration of oxygen (oximetry) byelectron paramagnetic resonance (EPR) spectroscopy are reported. Theprobe is synthesized as a needle-shaped microcrystalline particulate.The particulate shows a single-line EPR spectrum that is highlyexchange-narrowed with a line-width of 210 mG. The EPR line-width issensitive to molecular oxygen showing a linear relationship between theline-width and concentration of oxygen (pO₂) with a sensitivity of 8.5mG/mmHg. We studied a variety of physicochemical and biologicalproperties of LiNc-BuO particulates to evaluate the suitability of theprobe for in vivo oximetry. The probe is unaffected by biologicaloxidoreductants, stable in tissues for several months, and can besuccessfully internalized in cells. We used this probe to monitorchanges in concentration of oxygen in the normal muscle and RIF-1 tumortissue of mice as a function of tumor growth. The data showed a rapiddecrease in the tumor pO₂ with increase of tumor volume. Human arterialsmooth muscle cells, upon internalization of the LiNc-BuO probe, showeda marked oxygen gradient across the cell membrane. In summary, the newlysynthesized octa-n-butoxy derivative of lithium naphthalocyanine hasunique properties that are useful for determining oxygen concentrationin chemical and biological systems by EPR spectroscopy and also formagnetic tagging of cells.

Aerobic life relies on oxygen for respiration and bioenergeticmetabolism. In animals, especially mammals, under normal physiologicalconditions, oxygen delivery by blood to the tissues and tissueoxygenation are tightly regulated to maintain a balance [65], which isaltered during many pathophysiological states. Therefore, an accurateand a reliable method to determine its concentration in biologicalsystems is highly critical. Although several existing methods areutilized to measure oxygen concentration in absolute units or in somerelated parameter, a suitable technique for noninvasive and repeatedmeasurements of oxygen in the same tissue or cells on a temporal scaleis warranted. While electrode techniques have evolved as the standardmethods for measurement of oxygen, they generate analytical artifactsduring assay procedures at the freshly probed sites [66]. Near-infraredand magnetic resonance techniques such as nuclear magnetic resonance,blood oxygen level-dependent magnetic resonance imaging,Overhauser-enhanced magnetic resonance imaging, etc, on the other hand,are noninvasive methods, but they do not report usually the absolutevalues of oxygen concentration and lack the resolution of oxygenmeasurements [67-75]. Electron paramagnetic resonance (EPR), closelyrelated to the aforementioned magnetic resonance techniques, enablesreliable and accurate measurements of concentrations of oxygen [76]. TheEPR technique requires the incorporation of an ‘oxygen-sensing’paramagnetic spin probe into the system of interest. Two types of probesare used: (i) soluble probes that report the concentration of dissolvedoxygen and (ii) particulate probes that measure partial pressure ofoxygen (pO₂) in the milieu. Considerable progress has been made in thedevelopment and use of both types of probes [77-80]. The advantages inusing the particulate probe are higher resolution and their suitabilityfor repeated measurements in vivo without reintroduction of the probeinto the tissue. Both the naturally occurring and synthetic materialshave been useful for EPR oximetry [78, 79, 81-83].

Earlier, alkali metal derivatives of phthalocyanines such as lithiumphthalocyanine (LiPc) [77, 81, 84-86] and lithium naphthalocyanine(LiNc) [78, 87] were synthesized and their properties were studied indetail. The materials were characterized as crystalline solids composedof stacks of neutral free radical molecules [88]. The crystalline solidsexhibit a highly exchange-narrowed single line EPR spectrum, whose widthis sensitive to the partial pressure of molecular oxygen in theenvironment. Our interest in the utilization of oxygensensing radicalprobes for biological applications has lead to the synthesis anddevelopment of novel crystalline particulate materials with remarkableoxygen sensitivity and biocompatibility [77, 78, 85-87]. While we haveidentified that these materials enable us to perform accurate andrepeated measurements of oxygen concentration in tissues, we realizethat these particulate probes can very well be used in otherphysiological and biochemical studies that involve oxygen metabolism.The unique stability and paramagnetic property of these particulates canbe exploited by internalizing them into cells and in specific tissues tovisualize cell proliferation, migration and trafficking, as it isstudied using superparamagnetic particulates in magnetic resonanceimaging technology [89-91]. The EPR technique is advantageous inoffering high sensitivity and direct detection of the particulates andreporting the absolute value of oxygen concentration in the environment.We envision that this creates a multitude of applications in cell-basedtherapies and tissue engineering [92, 93].

In order to qualify as an ideal spin probe for targeted measurements incells and tissues, a paramagnetic particulate has to satisfy thefollowing criteria: (i) high spin density with a simple EPR absorptionpeak, preferably a single and sharp line (ii) long-term stability incells and tissues, maintaining its line-shape and oxygen sensitivity(iii) non-toxicity to the host cell or tissue (iv) ability to prepareparticulates of various sizes, and (v) ability to encapsulate in shellsor coating to enable attachment of other probes such as fluorescentlabels. Although a variety of paramagnetic spin particulates, includingnatural [82, 94] and semisynthetic [83, 95, 96] has been reported to beuseful as oximetry probes, they do not satisfy most of the aboverequirements. Hence, we focused our efforts on synthetic molecularcrystalline particulates, whose properties can be controlled andsystematically altered by appropriate molecular designs [77, 78, 85-87].In this manuscript, we report the synthesis, characterization andapplication of a new paramagnetic particulate spin probe. The probe is alithiated form of octa-n-butoxynaphthalocyanine neutral radical (FIG. 1)which is obtained in a microcrystalline form. The preliminary resultsindicate that the probe is useful for determining oxygen concentrationin chemical and biological systems by EPR spectroscopy and that it maysignificantly expand the capability of EPR oximetry.

Materials and Methods:

Lithium granules,5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine (Nc-BuO),n-pentanol, n-hexane, tetrahydrofuran and tert-butyl methyl ether wereobtained from Aldrich Chemical Co (St. Louis, Mo.). Minimal essentialmedium (MEM), Dulbecco's modified Eagle medium (DMEM), fetal bovineserum (FBS), glutamate and antibiotic (penicillin-streptomycin) werepurchased from Invitrogen, San Diego, Calif. Alamar Blue solution waspurchased from Biosource International (Camarillo, Calif.).

Synthesis of Lithium5,9,14,18,23,27,32,36-Octa-n-butoxy-2,3-naphthalocyanine (LiNc-BuO)Radical

Lithium granules (0.0053 g, 0.774 mmol) were added to n-pentanol (15 ml)and refluxed for 30 min under nitrogen atmosphere. The mixture wascooled down to room temperature and Nc-BuO (0.1 g, 0.0774 mmol) wasadded and refluxed gently for 2.5 h under nitrogen atmosphere. Aftercooling down to room temperature, 300 ml of tert-butyl methyl ether wasadded and filtered through a small silica gel plug. The solvent wasevaporated under reduced pressure to 3 ml of solution. The concentratewas dissolved in 100 ml of n-hexane. The greenish solution was slowlyevaporated under reduced pressure to yield shiny crystals of lithium5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine. The crystalswere washed with methanol and dried under vacuum. The yield was 81%.Microanalysis of the product was in good agreement with the formulaC80H88N808Li (Calculated: C, 74.1; H, 6.84; N, 8.6; Li, 0.53 Found: C,73.9; H, 7.12; N, 7.89; Li, 0.55).

Physicochemical Characterizations:

Electronic absorption spectra were measured in tetrahydrofuran solventusing a Cary 300 BIO UV-Visible Spectrophotometer. X-ray diffractionmeasurements were performed using a Bruker D8 Advance model X-raydiffractometer operating at 40 kV and 50 mA with Cu K1 á radiation(ë=1.5406 Å) using a Braun position-sensitive detector.

EPR Measurements:

EPR measurements were performed using a Bruker ER-300 spectrometeroperating at X-band (9.78 GHz). The spectral acquisitions were carriedout using custom-developed software (SPEX). Unless mentioned otherwise,the EPR line-widths reported are peak-topeak width (.Bpp) of the firstderivative spectra. The EPR line-width versus partial pressure of oxygencalibration curve was constructed from X- and L-band EPR measurements onLiNc-BuO equilibrated with oxygen/nitrogen gas mixture as reportedpreviously [78].

Animal Studies:

Female C3H mice were used in the present work. The mice were suppliedthrough the Frederick Cancer Research Center Animal Production,Frederick, Md. The animals were received at 6 weeks of age and housedfive per cage in climate-controlled rooms and allowed food and acidifiedwater ad lib. The animals were on average 50 days old at the time ofexperimentation and weighed 25±3 g. Experiments were conducted accordingto the principles outlined in the Guide for the Care and Use ofLaboratory Animals prepared by the Institute of Laboratory AnimalResources, National Research Council.

RIF-I Tumor Growth:

Radiation-induced fibrosarcoma (RIF-1) tumor cells, grown in monolayered culture, were injected subcutaneously in the right hind leg witha single cell suspension of 106 cells in 0.1 ml PBS. The animals wereobserved closely and the tumors became palpable approximately 5 daysafter injection.

Implantation of LiNc-BuO in Tumor and Gastrocnemius Muscle of Mice:

Mice were anesthetized with breathing of isoflurane (1.5%)-air mixturedelivered through a nose cone. About 10 μg of LiNc-BuO in the form ofmicrocrystalline powder (particulate size 5-20 μm) was implanted in thetumor (right leg) or gastrocnemius muscle of normal leg (left), using a21-gauge needle loaded with the particulate in the tip, and a wirestylus. The material was deposited at desired locations of the tissue byinserting the needle and then pulling it back, but keeping the wirestylus stationary and then removing the stylus from the tissue. In thecase of tumors, the material was carefully implanted at the center ofthe tumor at about 3 mm depth. The site of the probe insertion wasmarked with a permanent marker for convenient preparation of the animalfor repeated measurements. All the in vivo EPR measurements of LiNc-BuOin mice were made at least 24 h after the implantation.

In Vivo EPR Measurements in Mice:

The EPR measurements were carried out on anesthetized mice using L-band(1.32 GHz) spectrometer and a topical (surface loop) resonator asdescribed [33]. A plastic bedplate with a circular observation window(20 mm diameter) was used to rest the animal on the resonator. Theanimal was placed on the bedplate so that the observation spot wascentered at the slot. The animal was secured to the bedplate withadhesive tape and placed on top of the resonator so that the tumor orthe normal muscle was in direct contact with the active surface of theresonator. Anesthesia was maintained during the measurements withcontinuous delivery of 1.5% isoflurane mixed with air using a veterinaryanesthesia system (Vasco Anesthesia, Pro Tech Medical Inc., Hazel Crest,Ill.). The flow rate of the breathing gas mixture was maintained at 2L/min. The gas and anesthesia were delivered to the animal through anose cone and the excess air was removed through proper ventilationmaintaining the atmospheric pressure (760 mmHg). A thermistor rectalprobe was used to monitor body temperature. The body temperature wasmaintained at 37±1° C. using an infrared lamp.

Culture of Smooth Muscle Cells:

Human arterial smooth muscle cells (HASMCs) were obtained fromClonetics, San Diego, Calif. at passage 4. Cells were cultured andpassed in Ham's medium containing 225 ml of DMEM, 225 ml of F-12 medium,50 ml of FBS (10%), 5 ml of glutamate and 5 ml of antibiotics in a finalvolume of 500 ml. Cell cultures were maintained at 37° C. under 95%air/5% CO2 environment in 35 mm dishes. HASMCs up to passage 11 wereused in the current study.

Preparation of Particulates for Cell Culture Studies:

Fine crystals of freshly synthesized LiNc-BuO were suspended in Ham'smedium (10 mg/0.5 ml) and sonicated ten times for 30 sec on ice with aprobe sonicator at a setting of 5. The particulate mixture was cooled onice for 1 min between two successive 30 sec burst of sonication. At theend of the final round of sonication, the suspension was placed on icefor exactly 2 min to allow the heavier particulates to settle down atthe bottom of the tube and the decanted liquid was transferred to aseparate tube, which contained fine particulates of LiNc-BuO forintracellular delivery. The size of the particulates for intracellulardelivery was <2 μm as determined by optical microscopy.

Internalization of Particulates into Smooth Muscle Cells:

HMSMCs, at 70% confluence (104 cells/dish), in 1 ml Ham's medium weretreated with 50 μm of LiNc-BuO particulate suspension that containedparticulates of <2 μm prepared by the procedure as mentioned before. Thecells were maintained at 37° C. under 95% air/5% CO2 environment. At 6 hintervals, for 72 h, cells were examined under light microscope forinternalization of LiNc-BuO particulates. Upon confirming theparticulate uptake by all the cells in a given dish, the cells werewashed 12 times with ice-cold DMEM to remove unincorporated andextraneous particulates by gentle swirling and aspiration, scrapped in 1ml of DMEM, and centrifuged at 1000×g in a microfuge for 10 min. Theresulting cell pellet was gently resuspended in DMEM containing glucose(0.5 g/500 mL) for EPR analysis. Cells after LiNc-BuO internalizationand repeated washings were photographed under an inverted microscopewhile still adherent to the substratum of the 35 mm dish. Cell viabilitywas assessed by light microscopy and Alamar Blue assay according to themanufacturer's recommendations.

Results:

Physicochemical Characterization LiNc-BuO was synthesized as dark-greenneedle-shaped crystals of varying sizes, typically with 1-5 μm diameterand 5-50 μm length. The crystals were insoluble in water, but soluble inchloroform, dichloromethane, tetrahydrofuran, toluene, benzene andxylenes giving rise to green-colored solution. The crystals were stablein air at ambient conditions. The UV-visible absorption spectrum ofLiNc-BuO solution in tetrahydrofuran showed strong Q-bands at 857 and705 nm and a weak split Soret band at 449 nm, while the Nc-BuO (themacro cyclic ligand without Li) showed a strong Q-band at 865 nm. X-raydiffraction pattern showed strong diffraction peaks suggestive of a highdegree of crystallinity. A thorough investigation of the optical,magnetic, and structural property of LiNc-BuO will be publishedelsewhere [34]. Most of the studies were performed on fine crystals ofLiNc-BuO, with <2 μm in size, obtained by sonication of the originallysynthesized material in PBS or Ham's medium containing 10% fetal bovineserum. This was done to achieve particulates of uniform size forinternalization in cells and tissues. We used the term ‘particulates’ torefer to these crystals throughout the manuscript.

EPR Properties

The LiNc-BuO particulates exhibit a single-line EPR spectrum at roomtemperature (FIG. 2). The peak-to-peak width of the spectrum was highlydependent on the oxygen concentration of environment: 210 mG underanoxic (0% oxygen) conditions, 1550 mG in room air (20.9% oxygen or 159mmHg) and 6675 mG in 100% oxygen at 1 atmospheric pressure (760 mmHg).The shape of the spectrum was 100% Lorentzian. This is evidenced fromthe very good agreement between the experimental and simulated spectrain FIG. 2A and the random noise in the difference spectrum (FIG. 2B).The spin density, measured in comparison with diphenylpicrylhydrazyl(DPPH) radical, was 7.2×1020 spins/g. Microwave power saturation studiesof LiNc-BuO were performed to establish the useable range of powerlevels. It was observed that spectrum was not saturable for up to 25 mWat the X-band frequency. This shows that up to 25 mW microwave power canbe applied to enhance the signal intensity without compromising theoxygen sensitivity. Both the line-width and lineshape of theparticulates were not affected in aqueous dispersion media (water, PBS,cell culture media). However, no EPR spectrum was observed in organicsolvents, in which the compound is freely soluble, suggesting that theradical is not stable in the molecular form.

Effect of Molecular Oxygen:

The peak-to-peak width of the EPR spectrum of LiNc-BuO particulates wassensitive to oxygen concentration of the environment. The spectrum wasbroadened and its amplitude decreased in presence of oxygen. Theoxygen-dependent broadening of the EPR spectrum has been observed withLiPc, LiNc and several other paramagnetic solids [77-79, 85-87, 99-101].It is generally considered that the broadening in presence of molecularoxygen is due to the Heisenberg spin exchange between the radical andmolecular oxygen and results in shortening of the spin-spin relaxationtime T2. A plot of line-width measured as a function of oxygen partialpressure is shown in FIG. 3. It is observed that the line-widthincreases linearly with pO₂ in the range 0 to 760 mmHg suggesting thatthe spin exchange increases linearly with pO₂. The slope of theline-width versus pO₂ curve, which reflects the oxygen sensitivity ofthe probe line-width, is 8.5 mG/mmHg.

Effect of Biological Oxidoreductants, pH, Temperature, and Radiation:

Since our goal was to use the newly synthesized particulate material forbiological applications, we thoroughly investigated the EPR stability(paramagnetism) as well as sensitivity of the EPR line-width tomolecular oxygen in presence of a variety of biological oxidants,reductants, pH and radiation. The particulates when exposed tosuperoxide (generated by 0.2 mM xanthine+0.01 D/ml xanthine oxidase),hydroxyl (generated by 0.1 mM Fe2++1 mM H₂O₂), hydrogen peroxide (1 mM),singlet oxygen (generated by 1 mM Rose Bengal+light), alkylperoxyl(generated by aerobic decomposition of 10 mM AAPH at 37° C.), and nitricoxide (generated by 1 mM SNAP), GSH (10 mM) and ascorbate (5 mM) for 30min did not show any effect on the EPR spectrum or oxygen response. Wealso observed that pH of the medium in the range 2-10 had no effect ontheir EPR sensitivity to oxygen. We also treated the particulates with15.5 Gy of Cobalt-60 ã-ray irradiation for 10 min and found no effect onthe EPR properties of the particulates. These results suggest that theLiNc-BuO particulates are usable in a variety of extreme biologicalenvironments without any adverse effect on the integrity of data.

Stability in Tissues:

In order to evaluate the long-term stability of these particulates intissues, we implanted the particulates in the gastrocnemius muscletissue of mice and performed repeated measurements of pO₂ in the sameanimals over a period of time. About 10 μg of LiNc-BuO microcrystallinepowder was implanted in the gastrocnemius muscle of the right leg of C3Hmice (N=6). The EPR spectrum of the particulate in the leg was recordedperiodically up to 180 days following the implantation of particulate(FIG. 4). In order to verify the response of the particulate to oxygen,blood flow to the leg was constricted by gentle tying down of the upperleg for 10 min with an elastic band and the EPR measurement wasrepeated. Sharpening of the EPR spectral width during interruption ofblood flow to the leg was used as an indication of reduced tissueoxygenation and responsiveness of the particulate to changes in tissuepO₂. The pO₂ of the tissue under normal blood flow conditions was19.6±2.1 mmHg, while that of constricted tissue was 3.5±0.9 mmHg duringthe 180 day period. The non-zero pO₂ values in the flow-constrictedtissue suggest that the constrictions were not totally effective. Thiswas due to our efforts to make the measurements for at least 6 monthsand so deliberately avoided inflicting any permanent injury to thetissue while constriction. Mice were periodically sacrificed through the180 day study period and tissue pO₂ values were assessed to verify theregistration of anoxic pO₂ in the dead tissue. The pO₂ values in thedead tissues were close to zero. We also observed that the oxygensensitivity of the recovered particulates from the dead tissue was notchanged and was similar to that of the original unimplanted particulate.

Time-Response to Changes in Oxygenation:

The response time and reproducibility of the effect of O₂ in successivemeasurements were evaluated. The response of probe was measured from thechange in the EPR amplitudes to cycles of rapid switching of theequilibrating gases between nitrogen and room air as describedpreviously [78]. It was observed that the response was reasonably quickwith oxygenation occurring at ˜1 sec, while deoxygenation at ˜20 sec.Similar values of response time and amplitude were observed onsuccessive cycles of switching of gas. Thus, the experiments not onlyconfirm that the probe responds quickly to oxygen and offers steadyreproducibility, but also that oxygen is not irreversibly adsorbed andthat the absorption/desorption process is very rapid and reversible.Thus, the probe apparently is capable of responding to changes in oxygenconcentration almost instantaneously. A similar observation has alsobeen reported in the case of LiPc and LiNc crystals [78].

Measurement of Tumor pO₂ as a Function of Tumor Growth:

The LiNc-BuO particulate was implanted in the tumor on day 5 afterinoculation with tumor cells. The average tumor size at the time ofimplantation of the particulate was 6 mm in diameter. A similarimplantation of the particulate, as a control, was performed in thenormal muscle on the left leg of the same tumor-bearing mice. Tissue pO₂measurements were taken 24 h following implantation to avoid artifactsassociated with trauma and tissue injury caused by the particulateimplantation procedure. Measurements were performed in the tumor on theright leg and in the normal muscle on the left leg of each animal dailyfor the following 8 days. The mean pO₂ values from the tumor and muscletissue in 7 tumor-bearing mice are shown in FIG. 5. It was observed thatwhile the pO₂ in the muscle tissue (control) of the RIF-1 tumor-bearingmice remained constant during the study period (17.6±2.5 mmHg), thetumor pO₂ showed a continuous decrease towards hypoxia (<4 mmHg). It wasalso observed that the RIF-1 tumor showed an accelerated growth duringthe same period suggesting that the decrease in tumor oxygenation may berelated to tumor progression. The measurements were discontinued beyondday 8 as the tumors were too big (>20 mm in diameter) and continued tobe hypoxic with pO₂ levels <4 mmHg.

Intracellular Internalization of Particulates:

The light microscopy clearly showed that within 18 h of treatment ofHASMCs with LiNc-BuO particulates (<2 μm) in Ham's medium, almost allthe cells in the 35 mm dish internalized the particulates. There was noapparent cytotoxicity of the particulates up to 72 h following theirinternalization as revealed by the light microscopy and Alamar Bluecytotoxicity assay (data not shown).

Measurement of Intracellular pO₂ in Smooth Muscle Cells:

The particulates obtained by sonication of the LiNc-BuO crystals inHam's complete medium containing 10% fetal bovine serum wereinternalized into cells. The extracellular uninternalized particulateswere removed by repeated washings with medium. FIG. 6 shows a photographof cells internalized with the particulates. The intracellular oxygenconcentration, measured from the internalized particulates, was 142±2mmHg, while the extracellular pO₂ was measured to be 158±3 mmHg usingunsonicated particulates that were added to control cells (withoutinternalized particulates) prior to measurement. The data show that theparticulates are capable of reporting exclusively intracellular pO₂ wheninternalized into the cells.

Discussion:

The LiNc-BuO belongs to a new class of crystalline internal spin probe(CRISP) that has several advantages over the previously reportedparticulate probes, namely lithium phthalocyanine [77, 81] and lithiumnaphthalocyanine [78]. Although LiNc-BuO is a derivative of the othertwo, closely similar in molecular structure, its properties are verydifferent from its predecessors. Some of the distinct and advantageousfeatures of LiNc-BuO paramagnetic spin particulates are: (i) theirability to give rise to a single, sharp and isotropic EPR spectrumcharacterized by 100% Lorentzian shape obtained from crystalline powder(ii) their relatively very high spin density compared to LiPc or LiNc(iii) they exhibit a linear variation of line-width with pO₂ that isindependent of particulate size (iv) their long term stability intissues and (v) their ability to internalize in cells. In addition theLiNc-BuO particulates also show typical auto-fluorescence propertieswhich are under investigation. A complete three-dimensional X-raystructure elucidation of the crystals is in progress.

The anoxic line-width of LiNc-BuO is 210 mG. This is larger whencompared to that of LiPc, which we have reported to be <20 mG [77, 81].However, the value is smaller than that of LiNc (510 mG) though the LiNcis structurally closer to the LiNc-BuO [78]. This difference in theanoxic line-width of LiNc-BuO may be attributed to changes in theexchange interaction caused by the introduction of alkoxy substituentsto the naphthalocyanine macrocycle. On the other hand, the oxygensensitivity of LiNc-BuO (8.5 mG/mmHg) is much closer to that of LiPc(8.9 mG/mmHg) than that of LiNc (28.5 mg/mmHg). These differences inproperties among the particulates suggest that even a small change inthe structure can result in substantial change in the electron exchangeand oximetry properties.

The apparent spin density of LiNc-BuO (7.2×1020 spins/g), measured incomparison with diphenylpicrylhydrazyl (DPPH) radical, is seven-foldhigher than that of LiPc and comparable to that of LiNc (6.8×1020spins/g). The observed spin density of LiNc-BuO, only a relative value,is determined by comparing the intensities of the EPR spectra ofLiNc-BuO with DPPH measured under identical experimental conditions.However, the nature of spin dynamics could affect the absolute value ofspin density in the system as discussed for LiNc [78].

The oxygen-dependent broadening of the EPR spectrum has been observedwith LiPc, LiNc and several other paramagnetic solids [77-79, 85-87,99-101]. The broadening in presence of molecular oxygen is generallyattributed to the Heisenberg spin exchange between the radical andmolecular oxygen and results in shortening of the spin-spin relaxationtime T2 [102]. Alternatively, as per the mechanism proposed recently forLiPc microcrystalline powders [86], the O₂ can trap the self-exchangingor diffusing spins resulting in broad EPR lines. The latter mechanism ismore probable especially for the solid spin probes with self-interactingspins.

An important drawback with many particulate oximetry probes was theinstability of the probe in live tissues for prolonged periods of time.The most widely used LiPc particulate is stable in the gastrocnemiusmuscle tissue of mice for only about 3 weeks, beyond which the probeapparently looses its sensitivity to oxygen. Though the LiNc probe hasseveral other advantages over LiPc, its stability in tissue was limitedto only a few days. There were intense efforts to enhance their tissuestability over longer periods of time, but however, there has been nosignificant success to date [99, 103, 104]. Thus, it is important tonote that LiNc-BuO has tissue stability for 6 months, and appears tolast longer.

We have demonstrated the usefulness of the probe for making repeated andnoninvasive measurements in a RIF-1 murine tumor model. The data showthat the pO₂ levels in the normal (nontumor-bearing) leg muscle are moreor less constant while the values in the tumor of the same set ofanimals progressively decreased to hypoxic levels during the measurementperiod. It is particularly important to note that there are relativelysmall variations in the pO₂ between the tumors as seen from the dataanalysis. This observation suggests that the oxygen concentration in theRIF-1 tumor decreases as a function of tumor growth.

Though the implantation of the particulates into the tissue is invasive,it differs from other routine invasive techniques in many ways. Forexample, in the case of the commonly used Eppendorf electrode technique,the electrode is inserted at each sampling time during the measurementcausing local tissue injury and the pO₂ readings are obtained each timefrom the freshly injured site. Although in the case of particulateprobes, the implantation procedure is invasive, the measurements areperformed for days following the implantation of the particulate probeat the tissue sites where wound healing occurs after preparativesurgery. Furthermore, the implanted probe can be used repeatedly, aslong as the probe is stable and responsive to oxygen, without repeatedinsertions and surgery. Thus, the EPR oximetry technique is minimallyinvasive in terms of the requirement of one time implantation andsurgery and thus enables subsequent noninvasive measurement ofconcentration oxygen from the same location.

The advantage of LiNc-BuO is the ability to make particulates ofnanometer size without compromising its EPR and oxygen-sensingabilities. The smaller particulates can be internalized in a variety ofcells for different applications. For example, intracellular pO₂ can bemeasured from single cells. It is also possible to tag cells with theEPR particulate probes and study their migration, infiltration andproliferation over a period of time using EPR or MRI technologies, invivo. This will be similar to the capabilities of ultra smallsuperparamagnetic particulates of iron oxide (USPIO) that have beenactively pursued as contrast agents in magnetic resonance imaging[105-107]. The crystalline internal spin probe (CRISP) technology hasbiomedical applications including tissue repair, wound healing andoximetry, where in vivo EPR spectroscopy and imaging can be used. TheEPR spectroscopy has the advantage of direct detection of theseparticulates, compared to the contrast-based detection by MRI, as wellas the capability of measuring absolute concentration of oxygenconcentration in cells and tissues.

Summary and Conclusions:

A new butoxy-substituted naphthalocyanine-based radical probe, LiNc-BuO,with striking EPR properties was synthesized as fine crystals andcharacterized with significantly high spin density. The probe showed ahighly exchange-narrowed single line EPR spectrum that was sensitive tothe surrounding oxygen concentration. The effect of molecular oxygen onthe EPR line width was linear for up to 760 mmHg and highly reproducibleon successive applications, suggesting that it can be used as a probefor EPR-based oximetry application. The probe has definitive advantagesover other EPR oximetry probes reported earlier. The EPR spectrum isnonsatuarable up to 25 mW microwave power levels, and hence the signalto noise ratio can be substantially improved by increasing microwavepower during measurements. The sensitivity of the EPR line-width tomolecular oxygen is 8.5 mG/mmHg which suggests that changes in pO₂ canbe measured with reasonable resolution (˜0.2 mmHg) using this probeunder the experimental conditions described in this work. The probeshows a linear response of its line-width to pO₂ up to 100% molecularoxygen (760 mmHg), thereby enabling the measurement of the pO₂ even inthe higher range while maintaining the sensitivity. The probe is stableagainst a variety of biological oxidoreductants, stable in tissues formore than 6 months, and can be readily internalized in cells. Thus thenew octa-nbutoxy derivative of LiNc has unique properties that may beuseful for determining oxygen concentration in chemical/biologicalsystems and for magnetic tagging of cells.

Measurement of Oxygen Consumption in Mouse Aortic Endothelial CellsUsing a Microparticulate Oximetry Probe:

The purpose of this study was to determine the rate of oxygenconsumption in mouse aortic endothelial cells (MAECs) and to determinethe effect of a variety of inhibitors and stimulators of oxygenconsumption measured by electron paramagnetic resonance (EPR)spectroscopy utilizing a new particulate oximetry probe. We havepreviously demonstrated that the octa-n-butoxy derivative ofnaphthalocyanine neutral radical (LiNc-BuO) enables accurate, preciseand reproducible measurements of pO₂ in cellular suspensions. In thecurrent study, we carried out measurements to provide an accuratedetermination of pO₂ in small volume with less number of cells (20,000cells) that has not been possible with other techniques. In order toestablish the reliability of this method, agents such as menadione,lipopolysaccharide (LPS), potassium cyanide, rotenone anddiphenyleneiodonium chloride (DPI) were used to modulate the oxygenconsumption rate in the cells. We observed an increase in oxygenconsumption by the cells upon treatment with menadione and LPS, whereastreatment with cyanide, rotenone and DPI inhibited oxygen consumption.This study clearly demonstrated the utilization of EPR spectrometry withLiNc-BuO probe for determination of oxygen concentration in culturedcells.

Introduction:

Cellular oxygenation and oxygen consumption rate (OCR) are importantphysiological and metabolic indicators of cellular function. Normalcellular function and homeostasis require a critical level of oxygenconcentration (measured as oxygen tension, pO₂) in the cells to providean adequate supply of oxygen for the mitochondrial oxidativephosphorylation process [108, 109]. However, when the cellular oxygenlevel is altered from the critical level, the cellular homeostasis isdisrupted leading to abnormalities in cell growth, differentiation andsurvival. It is established that too little of oxygen (hypoxia) can leadto the activation of certain enzymes such as NAD(P)H oxidase, whichresults in the generation of reactive oxygen species (ROS) [110]. It isfurther shown that too much of oxygen (hyperoxia) may also lead to thegeneration of ROS from the mitochondrial electron transport chain andother sources [111]. The ROS cause oxidative stress by oxidizing thecellular components and by ultimately altering their structure andfunction. Although cells have evolved numerous antioxidant defensemechanisms against ROS-induced oxidative stress, for example, usingsuperoxide dismutase, catalase, glutathione peroxidase, and vitamin E,the concentration of oxygen in cells must be carefully controlled bymaintaining a balance between the ROS and antioxidants. In fact, theconcentrations of oxygen must be regulated such that the energy needs ofthe cell via oxidative phosphorylation are adequately met without alarge excess of oxygen.

In view of the importance of critical oxygen concentration in cells foroperation of normal cellular events, methods capable of determining theoxygen concentration in cells and tissues are highly crucial. Althoughmany methods are available to measure oxygen concentration in cells,each method has its advantages and disadvantages, and no single methodis completely satisfactory for cellular studies [112]. Electronparamagnetic resonance (EPR)-based oxygen measurements (oximetry)coupled with particulate probes [113-118] have some unique advantagesover the other methods. The particulate EPR probes for oximetry have thefollowing advantages: (i) they report pO₂, which is a better parameterin a heterogeneous cellular system (ii) they do not consume oxygen (iii)they provide higher resolution at lower pO₂ and (iv) they are stable incells and tissues for repeated measurements of oxygen tensions withoutreintroduction of the probe. A variety of particulate probes possessmany of these desirable properties and thus are useful for several invitro and in vivo clinical applications [113-118]. Particularly,lithiated macro cycles of phthalocyanine and naphthalocyanine have beenextensively investigated [113, 114]. Recently, we synthesized andcharacterized octa-n-butoxy derivative of naphthalocyanine neutralradical (LiNc-BuO), an analog of phthalocyanine with extendedbenzoannulation [119]. The LiNc-BuO neutral radical (oximetry probe), asopposed to LiPc [113], fusinite [117], glucose char [118] etc., offersmarked advantages, especially with regards to low microwave powersaturation, linear response to concentration of oxygen, dynamicmeasurement range and higher spin density. Recently, we demonstrated theutilization of this material to measure oxygen concentration in intactcells and in vivo biological systems with greater stability (>6 monthsin the gastrocnemius muscle of mice) and reproducibility in aqueous andphysiological environments [119]. Our earlier studies demonstrated thatthe LiNc-BuO microcrystalline powder can provide accurate and repetitivemeasurements of oxygen concentrations in intact cells and tissues.

In order to demonstrate the accuracy and reliability of the EPR oximetryutilizing the LiNc-BuO probe, we studied the pO₂ and oxygen consumptionrate (OCR) in mouse aortic endothelial cells (MAECs). These cells can becultured easily and considered as an established model for endothelialcells that are widely used [120]. The reported OCRs in endothelial cellsrange from 0.13 nmol/min/106 cells [121] to 87.5 nmol/min/106 cells[122]. The disparity in results, however, may be due to several factorsincluding differences in the cell type, experimental conditions andchoice of method. It should be noted that cellular metabolism can varyprofoundly depending on the conditions of incubation, presence andabsence of serum, growth factors and hormones and type of cell line usedin the study.

The purpose of this study was to determine the basal OCR in MAECs of lowcell density by EPR spectroscopy utilizing the LiNc-BuO particulateoximetry probe that is capable of providing accurate and reliablemeasurements of pO₂ in cellular suspensions. We demonstrated suchmeasurements in a small volume (20 μl) with as few as 20,000 cells. Wealso investigated the reliability of the method by studying the effectof several stimulators and inhibitors of cellular oxygen consumption.Menadione, and lipopolysaccharides (LPS) were chosen as stimulators ofcellular respiration. Cyanide, rotenone and diphenyleneiodonium (DPI)were chosen as inhibitors of cellular respiration. Here, we clearlydemonstrated that oxygen consumption by endothelial cells can bemeasured accurately by EPR oximetry utilizing LiNc-BuO. The reliabilityof the method was established by examining the sensitivity of oxygenconsumption by MAECs to various metabolic stimulators and inhibitors.

Materials and Methods—Reagents:

Lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine(LiNc-BuO) was used as a probe for measuring oxygen concentration incellular suspensions using EPR spectroscopy. LiNc-BuO belongs to theclass of crystalline internal spin probe (CRISP) particulates that wehave recently synthesized for measuring oxygen concentration in cellularsuspensions and tissues [119]. Menadione, DPI, LPS (Escherichia coliO128:B12), rotenone and potassium cyanide were purchased from SigmaChemical Company (St. Louis, Mo.). Stock solutions (1 mM) of menadione,DPI, and rotenone were freshly prepared in dimethyl sulfoxide (DMSO) andused immediately. LPS was prepared as 1 mg/ml of sterile phosphatebuffered saline (PBS) and stored at −20° C. until use. KCN was preparedin distilled water and used immediately.

Endothelial Cells:

MAECs used in this study were provided by Dr. Robert Auerbach at theUniversity of Wisconsin, Madison, Wis. MAECs were cultured in DMEMcontaining 5% fetal bovine serum and antibiotics(penicillin-streptomycin) and maintained at 37° C. in a humidifiedatmosphere of 5% CO2/95% air. MAECs were grown to 90% confluence in T-75flasks [120]. Cells from the flasks were detached by gentle scrappingwith a Teflon cell scrapper along with the medium, cell density wasdetermined, centrifuged at 1000×g at 4° C. for 10 minutes, andresuspended in a desired volume of PBS supplemented with glucose (0.1%)by gentle mixing for oximetry studies. Cell separation by trypsinizationwas avoided to keep the cell morphology and function in tact. Cells werecultured up to 6 passages and used in all the studies.

Cell suspensions were then treated immediately with variousoxygen-modulating agents such as menadione, LPS, KCN, DPI and rotenonein aerobic conditions. After the addition of ˜5 μg of the oximetry probe(LiNc-BuO), cell suspensions were drawn into capillary tubes (20 μl),which was then sealed at both ends using Critoseal®. Care was taken toavoid entrapment of any air bubbles in side the capillary. Cellviability was assessed before and after the EPR measurements by Trypanblue exclusion method and found to be >95%.

EPR Measurements:

The EPR measurements were carried out using a Bruker X-band (9.8 GHz)spectrometer (Bruker Instruments, Karlshrue, Germany) equipped withTM110 cavity. The cavity was rotated 90° so that the capillary tubefilled with the cell suspension could be kept horizontally to avoidsettling down of the cells. EPR spectra were acquired usingcustom-developed data acquisition software (SPEX). Unless mentionedotherwise, the EPR line-widths reported are peak-to-peak widths (.Bpp)of the first derivative spectra.

Calibration of LiNc-BuO Oximetry:

The LiNc-BuO crystals were calibrated for EPR oximetry as follows: Asmall amount (˜10 μg) of the probe was encapsulated in a 0.8 mm diametergas-permeable Teflon tube (Zeus Industrial Products, Orangeburg, S.C.,USA), both ends of the tube were sealed and the tube was inserted into a3 mm quartz EPR tube with both the ends open. The EPR tube was placedinto the TM110 microwave cavity (X-band) at the center of the activevolume of the resonator. Premixed oxygen and nitrogen gases of knowncomposition were flown through the EPR tube attached to a gas flow meter(Cole-Parmer, Vernon Hills, Ill., USA) and gas impermeable silicontubing (NOX, Wilmad Lab Glasses, Buena, N.J.). All measurements werecarried out after equilibrating the sample with the gas mixture 5 min.The flow rate of the gas mixture was maintained at 2 L/min. The totalpressure inside the EPR tube was maintained at 760 mmHg (atmosphericpressure) by exposing the other end of the tube to the ambientatmosphere. A linear variation of line-width was observed as a functionof partial pressure of oxygen (pO₂) as shown in FIG. 7.

Oxygen Consumption Measurements:

The OCRs were determined from pO₂ data as a function of time obtainedfrom cell suspensions in a sealed capillary tube. The followingexpression was used to calculate the OCR and expressed as nmol/min/1×106cells: OCR=m.á, where m is the slope of the pO₂ curve (in mmHg/min) anda is the solubility of oxygen in water (1.59 nmol/mmHg at 22° C.). OCRwas expressed as nanomoles of oxygen/min/106 cells.

Data Analysis:

All values are expressed as means±SD of 4 to 6 independent experiments.ANOVA and student's t-test were used for statistical analysis.Differences between groups were considered to be significant at P<0.05.

Results—Effect of Molecular Oxygen on the EPR Spectrum of LiNc-BuO:

The effect of molecular oxygen (O₂) on the EPR spectrum of LiNc-BuO isshown in FIG. 7. The LiNc-BuO exhibited a single EPR peak withpeak-to-peak width of 210 mG under anoxic condition. The spectrum wasbroadened with a concomitant decrease in amplitude in presence ofoxygen. FIG. 7 shows the width of the probe as a function of partialpressure of oxygen (pO₂). It was observed that the width increasedlinearly with pO₂ in the range 0-160 mmHg. The slope of the curve, whichreflected the oxygen sensitivity of the probe to oxygen, was 8.5mG/mmHg. Thus the probe apparently was capable of measuring oxygentension to ˜0.1 mmHg resolution in the physiological range.

Oxygen Consumption Measurements:

Typical time course data of pO₂ measured in suspensions of MAECs thatwere treated with various agents are shown in FIG. 8. The measurementswere performed routinely up to 20 min in a closed volume of 20 μl ofsuspension containing 20,000 cells. The data showed a linear decrease ofpO₂ over time in all cases. The OCR in untreated control cells was3.07±0.48 nmol/min/106 cells. Cells treated with menadione and LPSshowed increased rates of oxygen consumption while cells treated withDPI, KCN and rotenone showed complete inhibition of oxygen consumption.

Effect of Menadione:

Menadione is a redox-cycler that uncouples oxidative phosphorylation.The effect of menadione on the oxygen consumption by MAECs wasinvestigated in the dose range of 10-200 μM. The results are shown inFIG. 9. A significant increase in the OCR was observed when MAECs weretreated with menadione (10 and 50 μM) whereas a decrease in the OCR wasobserved upon treatment of cells with higher concentration of menadione(100 and 200 μM). A decrease in the OCR at the higher concentration ofmenadione might be due to enhanced production of oxygen radicals, whichapparently inhibited the mitochondrial respiration. An increase in theOCR when MAECs were treated with the lower concentrations of menadionemight be due to its redox-cycling activity and uncoupling of oxidativephosphorylation.

Effect of Lipopolysaccharide:

The effect of LPS (an endotoxin of bacterial origin) on the OCR in MAECswas studied (FIG. 10). Oxygen consumption by MAECs was measuredimmediately after treatment with LPS (10 and 20 μg/ml). As seen in FIG.10, an increase in the OCR was observed in cells treated with 10 and 20μg/ml of LPS. On the other hand, cells incubated for 2 h at 37° C. inpresence of 10 and 20 μg/ml of LPS showed a decrease in the OCR whichwas not statistically significant compared to untreated cells. Thus, itappears that the effect of LPS on the oxygen consumption by MAECs wasboth dose- and time-dependent.

Effect of Stimulators and Inhibitors on the Oxygen Consumption Rates:

FIG. 11 shows a comparison of the OCRs in MAECs with several stimulatorsand inhibitors of cellular respiration. As shown in the figure,menadione and LPS increased the OCRs. Measurements performed in presenceof inhibitors of mitochondrial respiration, namely, KCN (100 μM),rotenone (100 μM) and DPI (a flavoprotein inhibitor, 100 μM) indicatedthat there was a complete inhibition of the OCR in MAECs.

Discussion:

The results showed that the OCRs in MAECs can be measured accurately byEPR oximetry in a small volume suspension containing 20,000 cells. Thiswas feasible by the utilization of LiNc-BuO particulate oximetry probewhich has high sensitivity and higher resolution for the determinationof oxygen concentration. The high sensitivity of the EPR line-width ofLiNc-BuO (8.5 mG/mmHg) is particularly important for two reasons: (i)the oxygen consumption measurements in cell suspensions can be performedin a relatively shorter period (usually 10 min) as opposed to theelectrode or optical techniques which require several hours [123] (ii)measurements can be performed in small volumes (10-20 μL) and with lessnumber of cells, as has been demonstrated in the present work. Thus, anyalterations in the OCR due to higher cell densities or to exposure ofcells to varying concentration of oxygen for prolonged periods of timecan be eliminated by this method.

The OCR of untreated control MAECs measured at 22° C. was 3.07nmol/min/10⁶ cells. While this value is in the range of values reportedfor similar cells by other techniques, there are some markeddifferences. For example, James et al. [121] used 15N-PDT, a soluble EPRoximetry probe that measures average dissolved oxygen concentration inintra- and extracellular space of the porcine aortic ECs, and reportedan OCR of 0.13 nmol/min/106 cells. A higher OCR (1.45 nmol/min/106cells) was reported by Kjellstorm et al. [124] in the rat pulmonaryarterial endothelial cells using an oxyhemoglobin-basedmicrorespirometric method. They also observed comparable values in ECsfrom the human umbilical cord veins, but a significantly lower value inthe bovine aortic ECs (0.3 nmol/min/106 cells). Motterlini et al. [123]reported a value of 1.00 nmol/min/106 cells in the cultured vascular ECs(0.5×106 cells/ml) obtained from the porcine thoracic aorta using anoptical method based on the oxygen-dependent quenching of aphosphorescent probe. The disparity in the results might be due toseveral factors such as the differences in the origin of the cells,conditions of the incubation, as well as the presence or absence ofserum, growth factors and hormones. Further, the differences in thedetection techniques might also contribute to the measured values.

The rate of respiration in cells in suspension may depend on the ratioof oxygen to the cell density. Several studies have shown changes incellular metabolism parallel to changes in the oxygen concentration inthe suspension or to changes in the cell density [125]. The dependenceof the OCR on oxygen concentration in the medium is usually evidenced bynonlinearity in the oxygen versus time curve. Absence of any departurefrom linearity in the oxygen consumption curve (FIG. 8) suggested thatthe cellular respiration by the MAECs, at the cell density used, was notaltered due to depletion of oxygen in the medium. Motterlini et al.[123] observed that the OCR of the porcine aortic ECs was dependent onthe cell density, with the rate decreasing from 1.0 to 0.6 nmol/min/106cells when cell density was increased from 0.5-4×10⁶ cells/ml. However,we did not observe any significant change in the OCR of MAECs in therange of 0.5-4×10⁶ cells/ml (data not shown), suggesting that therespiration rate was not affected by cell density in this range.

Endothelial cells are generally characterized with lower OCR whencompared to other cells, e.g., smooth muscle cells, myocytes or Chinesehamster ovary cells. The lower consumption rate of the endothelial cellsis attributed to the presence of fewer number of mitochondria in thesecells [126]. The observation that cyanide (an inhibitor of complex I) ofthe electron transport chain) and rotenone (an inhibitor of complex I)completely inhibited the consumption of oxygen suggested that theobserved oxygen utilization in the untreated cells was primarily due tomitochondrial respiration. This is further confirmed by the inhibitoryeffect of DPI, which is a blocker of flavoprotein complex of NADPHoxidase and complex I of mitochondrial respiration.

Menadione is a redox cycler that uncouples oxidative phosphorylation inthe mitochondria leading to increased oxygen consumption. It is alsoknown to induce oxidative stress in cells by generating superoxide andother downstream oxidants in the mitochondria. This causes bothstructural and functional damage to mitochondria and membranes in cells.While the pro-oxidant activity of menadione may strongly depend on theintracellular oxygen availability, the deleterious effect of theoxidants leading to mitochondrial injury may impair cellularrespiration. The results of the present study clearly established theinvolvement of the OCR on the concentration of menadione. At lowerconcentrations of menadione an increase in the consumption of oxygen wasobserved. A two-fold increase in the OCR was measured at 50 μMconcentration of menadione. Since the measurements were performedimmediately after the addition of the quinone, the increase inconsumption might be attributed to the increased generation or inductionof reactive oxygen species in addition to the enhancement of oxidativephosphorylation in the mitochondria. At concentrations higher than 50μM, however, the OCRs were significantly less compared to the controlvalues, suggesting that the normal mitochondrial respiration might beimpaired due to damage caused by higher concentrations of the drug.

Endotoxemic sepsis is associated with inadequate tissue oxygenation andaltered distribution of oxygen in different organs [127]. Dysfunction ofvascular endothelium and consequent damage to vascular tissues areattributed to be the major determinants in organ dysfunction mediated byendotoxin. Several studies which investigated the possible impairment ofoxygen utilization in vascular cells treated with endotoxin showedconflicting results. A recent study by James et al. [121] showed thatthe influence of endotoxin on the rate of oxygen utilization is verymuch dependent on the cell type. While the CHO and kidney cortex cellsshowed markedly decreased oxygen consumption after treatment with LPS,ECs did not show any response to LPS. However, more recently Motterliniet al. [123] showed a 46% decrease in the rate of oxygen consumption inthe porcine aortic ECs that were exposed to 1 μg/ml LPS. Our results inMAECs, measured immediately after treatment with 10 μg/ml LPS, indicateda 34% increase in oxygen consumption. However, cells treated withsimilar concentration of LPS but measured after 2 h of incubation showeda 16% decrease in the OCR compared to untreated cells. The increase inconsumption of oxygen during the first 20 min after treatment with LPSmight be attributed to the oxidative burst of vascular NADPH oxidase.Recent studies have suggested that a phagocyte-type NADPH oxidase is asignificant source of intracellular ROS in cardiovascular cells [128].Pro inflammatory mediators such as TNF-á are known to stimulate NADPHoxidase in endothelial cells [129]. The oxygen consumption wascompletely blocked by DPI, a known inhibitor of NADPH oxidase suggestingthe LPS-induced oxidative burst in the ECs. We found a decrease in therate of oxygen consumption after 2 h of incubation with LPS. This mightbe due to the inhibition of mitochondrial respiration by nitric oxide.LPS is known to simulate NO from iNOS [130]. The time required for theendogenous stimulation of iNOS is usually 2-4 h. Nitric oxide canpotentially regulate cellular oxygen consumption by binding to theoxygen binding site of cytochrome oxidase, resulting in reversibleinhibition of mitochondrial respiration [131]. Higher concentrations ofNO or its derivatives like peroxynitrite can also cause irreversibleinhibition of respiration at multiple sites within mitochondria[132-134].

Summary and Conclusions:

Oxygen consumption rates of MAECs in suspension were determined usingEPR oximetry. The method utilized a microparticulate spin probe(LiNc-BuO) with a high sensitivity for oxygen, enabling accuratemeasurement of pO₂ in solution. We determined the effect of metabolicstimulators and inhibitors such as menadione, LPS, cyanide, rotenone,and DPI on the OCR. The measurements were performed in a volume of 20 μLcontaining 20,000 cells (cell density: 1×106 cells/ml) contained in aclosed capillary tube. A linear decrease in pO₂ was observed as afunction of time suggesting that the cellular respiration wasindependent of oxygen concentration in the medium. We observed anincrease in oxygen consumption when MAECs were treated with menadioneand LPS, whereas cyanide, rotenone and DPI inhibited oxygen consumption.In summary, we demonstrated that accurate measurements of cellularoxygen consumption can be performed in small volumes of cellularsuspensions using microparticulate-based EPR oximetry.

Simultaneous Measurement of Oxygenation in Intracellular andExtracellular Compartments of Lung Microvascular Endothelial Cells:

A new technique is described for simultaneous determination of intra-and extracellular oxygen concentrations (pO₂) in bovine lungmicrovascular endothelial cells (BLMVECs) using electron paramagneticresonance (EPR) oximetry. The method utilizes dual spin probes, oneexclusively internalized in cells and the other placed extracellularlywhich are capable of reporting oxygenation simultaneously from the twodistinct regions. The measurements were performed in BLMVEC suspensionsof 20 μL volume containing 4,000 cells. The extracellular pO₂ wasmeasured using a trityl EPR probe (TAM, 10 μM), a tricarboxylate anion,that stays exclusively in the extracellular space. The intracellularoxygen was measured using a pre-internalized particulate spin probe,lithium octa-n-butoxynaphthalocyanine (LiNc-BuO), which enables highlyaccurate and precise measurements of intracellular pO₂. Because there isa wide discrepancy in the reported values of cellular oxygenation by andlarge due to differences in the methods employed, we utilized the dualEPR probe technique to measure the oxygen gradient that apparentlyexists across the cell membrane. The intra- and extracellular pO₂ were139±2.5 and 157±3.6 mmHg, respectively, for cells exposed to room air(pO₂: 159 mmHg). A fairly smaller gradient of oxygen was observed incells exposed to 7.5% oxygen (pO₂: 57 mmHg). There was no significantdifference in the intra- and extracellular pO₂) when cells were treatedwith either menadione (50 μM) or cyanide (100 μM). In conclusion, thisstudy confirms the feasibility of simultaneous and accurate measurementsof intra-extracellular pO₂ using LiNc-BuO and TAM EPR oximetry probes.

Oxygen is an important modulator of cellular functions in both normalphysiology and disease states. Cells respond to oxygen over a wide rangeof concentrations from anoxia to hyperoxia. Baseline metabolism andfunction typically occur in normoxic environments (30-90 mmHg of O₂) andcan modulate differentiated cell functions [149]. Hyperoxic conditionsoften result in the generation of reactive oxygen species (ROS) thathave been implicated in cell injury via lipid peroxidation and cytokineexpression [139]. In lieu of such diversity in cellular responses tooxygen, the dynamics of tissue oxygenation, including the transport ofoxygen and possible existence of oxygen gradient across the cellmembrane needs to be measured accurately. Various methods such asmanometry, photometry, mass spectrometry and polarography (Clark-typeelectrochemical) have been described to measure concentration and uptakeof cellular oxygen [137, 138, 153, 159, 165]. The microelectrodetechnique, despite being used widely, has disadvantages as it consumesoxygen during measurement, apparently causes systematic error under verylow oxygen concentrations, requires insertion into the tissues, disturbsthe local environment and causes tissue damage [166, 167].

Although the determination of extracellular oxygen concentration in cellsuspensions is straightforward, the measurement of intracellular pO₂ iscomplicated. There are a few methods available to accomplish this, forexample, by insertion of an intracellular oxygen electrode into a singlecell [167, 168] or by fluorescence quenching by O₂ following thecellular uptake of fluorescent probe, pyrenebutyric or 2-nitroimidazole(EF5) [136, 151]. Electron paramagnetic resonance (EPR) spectroscopy,coupled with the use of oxygen-sensitive spin probes, has become apotential technique for accurate and precise determination of oxygenconcentrations in a variety of biological samples, including tissues andcells [141, 147, 154, 161, 170]. The technique, referred to as ‘EPRoximetry’, uses soluble molecular spin probes for the determination ofdissolved oxygen concentration and particulate spin probes for targeteddetermination of local oxygen tension (partial pressure of oxygen, pO₂)in tissues and cells [144]. The particulate probes have uniqueadvantages over the other EPR oximetry probes: (i) they report pO₂,which is a better parameter in a heterogeneous cellular system (ii) theydo not consume oxygen (iii) they provide higher resolution at lower pO₂and (iv) they possess greater stability in cells and tissues, so that,repeated measurements of oxygen tensions can be made for months withoutreintroduction of the probe. Hence, the particulate oximetryprobe-coupled EPR spectroscopic determination of oxygen has advantagesover the other methods of determination of oxygen in biological samples[137, 153, 159, 165]. A variety of particulate probes that possess manyof these desirable properties are useful in studies in vitro to in vivo[144, 152]. Recently, we synthesized and characterizedocta-n-butoxy-substituted naphthalocyanine neutral radical (LiNc-BuO),which exhibits marked advantages, especially with respect microwavepower saturation, linear response to concentration of oxygen, dynamicmeasurement range and higher spin density [156]. We have demonstratedthe application of this material by successfully internalizing into thelung microvascular endothelial cells in culture for measuringintracellular pO₂. The probe is capable of providing reliablemeasurements of intracellular pO₂ with 0.1 mmHg resolution and themeasurements can be made in a single cell.

The aim of the present study was to demonstrate the accuracy andreliability of the EPR oximetry method for simultaneous measurement ofintracellular pO₂ in bovine lung microvascular endothelial cells(BLMVECs) utilizing internalized particulates of LiNc-BuO andextracellular pO₂ using TAM. We have also measured the intracellular andextracellular pO₂ in these cells in presence of metabolic inhibitorssuch as menadione (50 μM) and potassium cyanide (100 μM). As we havepreviously demonstrated that the LiNc-BuO enables very accurate andreliable measurement of pO₂ in cellular suspensions, we envisioned thatthe measurements will provide accurate values of intracellular pO₂ thathave not been possible with the other techniques. Further, we extendedsuch measurements to smaller sample volume (20 μl) with 4,000 cells. Weobserved an intracellular pO₂ of 139 mmHg and extracellular pO₂ of 157mmHg with an oxygen gradient of 18 mmHg under aerobic conditions. Agradient of 9 mmHg of oxygen (extra and intracellular pO₂ were 64 and 55mmHg respectively) was observed when the cells were exposed to 7.5%oxygen. Menadione and potassium cyanide did not affect significantly theintra- and extracellular pO₂ levels.

Materials and Methods—Reagents:

Lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine(LiNc-BuO) was used as a probe for measuring intracellular oxygenconcentration by EPR spectroscopy. LiNc-BuO belongs to the class ofcrystalline internal spin probe (CRISP) particulates that we haverecently reported for measuring oxygen concentration in cellularsuspensions and tissues [156]. TAM was a gift from Nycomed Innovations(Malmo, Sweden). The EPR properties of TAM have been well characterized(1). Menadione and potassium cyanide were purchased from the SigmaChemical Company (St. Louis, Mo.). Stock solutions (1 mM) of menadioneand KCN were prepared freshly in dimethylsulfoxide and distilled water,respectively and used immediately. Minimum essential medium (MEM), fetalbovine serum and antibiotics were obtained from GIBCO-Invitrogen, Calif.

Bovine Lung Microvasular Endothelial Cells (BLMVECs) Culture:

The BLMVECs used in this study were obtained from the VEC Technologies,Inc, New York. BLMVECs cultured in MEM were maintained in 75 mm flasksat 37° C. in a humidified atmosphere of 5% CO₂/95% air and grown tocontact-inhibited mono layers with a typical cobblestone morphology[157]. Cells from each primary flask were detached with 0.05% trypsin,resuspended in fresh medium, and cultured in complete medium to 70%confluency for other studies. Cells from passages 10-14 were used in allthe experiments.

Preparation of Particulates for Cell Culture Studies:

Microcrystalline particulates of LiNc-BuO were suspended in complete MEMmedium (10 mg/0.5 ml) and sonicated for 30 sec pulse for ten times onice with a probe sonicator at a setting of 5. The particulate suspensionwas cooled for 1 min between two successive 30 sec bursts of sonication.At the end of sonication, the suspension was placed on ice for exactly 2min to allow the heavier particulates to settle down at the bottom ofthe tube and the supernatant liquid was transferred to a separate tubefor intracellular delivery. The solution contained fine particulates ofLiNc-BuO with a particulate size <2 μm. All the preparations werecarried out under sterile conditions.

Internalization of Particulates into Endothelial Cells:

BLMVECs, at 70% confluence (104 cells/35 mm dish), in 1 ml complete MEMwere treated with 50 μl of LiNc-BuO particulate suspension thatcontained particulates of <2 μm prepared by procedure as described underMaterials and Methods. The cells were maintained at 37° C. under 95%air/5% CO₂ environment. At 6 h intervals, for 72 h, cells were examinedunder light microscope for internalization of LiNc-BuO particulates.Upon confirming the particulate uptake by all the cells in a given dishafter 48 h of exposure to particulates, the cells were washed 12 timeswith ice-cold MEM to remove unincorporated and extraneous particulatesby gentle swirling and aspiration, scrapped in 1 ml of MEM, andcentrifuged at 1000×g in a microcentrifuge for 10 min. The cell pelletwas resuspended in MEM containing glucose (0.5 g per 500 ml), at adensity of 2×105 cells/ml and used for EPR analysis. Cells afterLiNc-BuO internalization and repeated washings were photographed underan inverted microscope while still adherent to the substratum of the 35mm dish. Cell viability was assessed by light microscopy and Alamar Blueassay according to the manufacturer's recommendations. Cell separationby trypsinization was avoided to keep the cell morphology and functionin tact.

A 20 μL volume of the cell suspension containing 4,000 cells and 10 μMTAM was drawn into a gas-permeable Teflon tube and subjected to EPRspectroscopy as described below. The measurements were also carried outwith Menadione (50 μM) and KCN (100 μM). Cell viability was assessedbefore and after the EPR measurements by Trypan blue exclusion methodand found to be >95%.

EPR Measurements:

The EPR measurements were carried out using a Bruker X-band (9.8 GHz)spectrometer (Bruker Instruments, Karlshrue, Germany) equipped withTM110 cavity. EPR spectral acquisitions were performed usingcustom-developed data acquisition software (SPEX) that was capable offully automated data acquisition and processing. Unless otherwisementioned, the EPR line-widths reported are peak-to-peak widths (.Bpp)of the first derivative spectra.

Calibration of LiN-BuO and TAM Oximetry:

The LiNc-BuO crystals were calibrated for EPR oximetry as describedearlier [156]. Measurements of the line width of LiNc-BuO afterequilibration with a series of oxygen and nitrogen gas mixtures wereperformed. Calibration was performed over the oxygen concentration range(0-21%) with oxygen/nitrogen mixtures. A linear variation of line-widthwas observed as a function of partial pressure of oxygen (pO₂) in theentire range of 0-160 mmHg. Similarly, the calibration of TAM (10 μM)was also performed using different oxygen concentrations (0-21%). Theoxygen-induced line-broadening (change in peak-peak width) of the signalwas used to measure extracellular oxygen concentration. The line shapeof the EPR signal was precisely simulated using a Lorentzian functionsand the Lorentzian width was used to establish the calibration curve.

Simultaneous Measurements of LiNc-BuO and TAM Line-Shapes:

LiNc-BuO and TAM were used as site specific oximetry probes to measureintra- and extracellular oxygen concentration, respectively. BothLiNc-BuO and TAM give a single-line EPR spectrum, whose amplitude(intensity) and width depend on the amount/concentration of the probeand oxygen, respectively. Since the g-factor of LiNc-BuO (g=2.0024) andTAM (g=2.0030) are slightly different, their spectra do not overlapcompletely and show a composite feature where the two peaks can beseparated by computer-simulation (FIG. 12).

The LiNc-BuO absorption profile is characterized by 100% Lorentzian[156], while the TAM signal can be approximated to be Lorentzian underconditions of oxygen-induced broadening. Thus, the deconvolutionrequires a simple two-component Lorentzian fitting to the measuredspectrum. We validated the faithfulness of the deconvolution byperforming the simulation under different combination of oxygenbroadening to the probes. The reproducibility was very good (R2>0.99)for non-zero oxygen concentrations. The line-shape of TAM wasnon-Lorentzian under anoxic conditions.

It should also be noted that there was no effect of TAM on the EPRspectrum of LiNc-BuO and vice versa, when the two probes were suspendedin the same medium having physical contact. This suggests that the twoprobes can be used together. However, in our experiments the probes weredistributed in different regions (intra- and extracellular) and hence,such a contact did not exist. The components were separated using PEAKFIT (SPSS, Chicago, Ill.) software and the intracellular andextracellular pO₂ were determined from the calibration curves ofLiNc-BuO and TAM.

Data Analysis:

All values are expressed as mean±SD of 4 to 6 independent experiments.ANOVA and student's t-test were used for statistical analysis.Differences between groups were considered to be significant at P<0.05.

Results—Internalization of LiNc-BuO Crystals into BLMVECs:

The light microscopy, as shown in FIG. 13, clearly showed within 18 h ofBLMVECs treatment with LiNc-BuO particulates (<2 μm) in complete MEM,nearly 95% of the cells internalized the particulates in a 35 mm dish.The internalized particulates show no cytotoxicity up to 72 h asevidenced by the light microscopy and Alamar Blue cytotoxicity assay. Atthe time of measurements, the viability of the cells that internalizedLiNc-BuO was >95% as studied with 0.4% Trypan blue exclusion method.

The mean spin density of the LiNc-BuO particulates internalized in asingle cell was calculated in comparison with a standard solution of TAMto be 6×10¹¹ spins/cell. This sensitivity is greater than that offeredby the X-band EPR spectrometer, which is typically better than 1×10¹⁰spins. Thus, one can measure EPR spectrum from a single cell that isinternalized with the LiNc-BuO particulates.

Effect of Molecular Oxygen on the EPR Spectrum of LiNc-BuO and TAM:

FIG. 14 shows the width of the probe as a function of partial pressureof oxygen (pO₂) in the range 0 to 158 mmHg. It is observed that thewidth increases linearly with pO₂ in the range 0-160 mmHg. The slope ofthe curve, which reflects the oxygen sensitivity of the probe to oxygen,is 8.5 mG/mmHg. Thus the probe is capable of measuring oxygen tension to˜0.1 mmHg resolution in the physiological range. Similarly, TAM exhibitsa peak-to-peak width of 146 mG under the anoxic condition and thespectrum is broadened in the presence of oxygen. The oxygen sensitivityof this radical is 0.36 mG/mmHg. The line shape of the EPR signalobtained with simultaneous use of intracellular LiNc-BuO andextracellular TAM was simulated precisely using two Lorentzian functionsand the Lorentzian width was used to measure pO₂ from the calibrationcurve.

Intra- and Extracellular Oxygen Concentrations

The internalized BLMVECs (4,000 cells) mixed with TAM (10 μM) in a 20 μLvolume of aerated solution, showed an oxygen gradient of 18 mmHg with anintracellular pO₂ of 139 mmHg and extracellular pO₂ of 157 mmHg (FIG.15). A gradient of 9 mmHg of oxygen was observed when cells were exposedto 7.5% oxygen. Internalization of LiNc-BuO particulates into BLMVECsand the feasibility of accurate measurement of intracellular pO₂ wereconfirmed in cell lysates prepared by brief sonication (5×10 sec) at 4°C. The pO₂ measured in the lysate was 158 mmHg. This observation clearlyindicated the internalization of particulate probe into BLMVECs and theexistence of oxygen gradient between intra- and extracellularcompartments. There was no significant change in pO₂ and oxygen gradientin cells treated with menadione (50 μM) or cyanide (100 μM) (FIG. 16).

Discussion:

Oxygen gradient in physiological systems plays an important role in bothmaintaining homeostasis and inducing cellular responses. Therefore, anaccurate and a reliable method to determine its concentrations in cellsand tissues is highly critical. Values of oxygen gradient in cellsmeasured by various methods reported so far in the literature varywidely range from 1 to 40 μM [140, 142, 148, 160, 162-164]. This broaddiscrepancy apparently is due to technical difficulty associated withaccurate measurement of intracellular oxygen concentration underphysiological conditions. Using nitroxides and other agents, several newmethods based on EPR oximetry technique have been developed to measureintracellular oxygen concentrations in cells [143, 145-147, 158]. Theparticulate probe-based EPR oximetry, used in the present study has manyadvantages over the other oximetry probe-based EPR spectroscopy andother widely used methods to measure intracellular pO₂. Some of thedistinct and advantageous features of LiNc-BuO paramagnetic spinparticulates are; their ability to give rise to single sharp andisotropic EPR spectrum characteristic with 100% Lorentzian shape, linearvariation of line-width with pO₂, that is independent of particulatesize and most importantly their ability to internalize in cells. We havetaken advantage of these favorable characteristics of LiNc-BuOparticulates, successfully internalized them into in BLMVECs andmeasured intracellular pO₂ using EPR spectroscopy.

The intra- and extracellular pO₂ measured by this technique in BLMVECswere 139 mmHg and 157 mmHg, respectively at room air with a gradient of18 mmHg. This technique also revealed the existence of a small oxygengradient of 9 mmHg at 7.5% (pO₂: 57 mmHg) oxygen (extra andintracellular pO₂ were 64 and 55 mmHg respectively). Similar finding wasalso observed by others [150]. This suggest that the cells possessdifferent gradients when exposed to different oxygen concentration and asmaller gradient exist at lower oxygen concentrations. Santini et al.[158] used fusinite as an EPR oximetry probe to measure intracellularmolecular oxygen in K56 (an erythroleukemic cell line) and A 431 (anepidermal carcinoma cell line) and demonstrated that menadione (200 μM)increased both intra- and extracellular pO₂ by 10-15%. But in our study,menadione (50 μM) did not alter intra and extracellular pO₂ in BLMVECs.This may be due to different experimental conditions, dose and differentcell types used. Khan et al. [150] measured intra- and extracellularoxygen concentrations in CHO cells by EPR oximetry using 15N-PDT andLiPc as intra- and extracellular probes, respectively. The extra- andintracellular oxygen concentrations observed in this study were 162 mmHg(1 μM of oxygen is equal to 0.714 mmHg in aqueous solution) and 129 mmHgat 150 mmHg and 38.5 and 34.2 mmHg at 35 mmHg of pO₂. Using thistechnique, they demonstrated that plasma membrane cholesterol is animportant barrier in regulating the oxygen gradient across the cellmembrane.

In our recent study, we used LiNc-BuO particulate probe and measured therate of oxygen consumption in mouse aortic endothelial cells in presenceof various stimulants and inhibitors of respiration [155] and alsomeasured the pO₂ in normal and tumor tissues [156]. In comparison withother oximetry probes, the unique advantage of LiNc-BuO is to prepareparticulates of nanometer size without compromising its EPR behavior andoxygen-sensing abilities. The smaller particulates can be internalizedin a variety of cells for different applications. It is possible tomeasure the pO₂ from a single cells internalized with LiNc-BuO.

The intracellular molecular oxygen is critical in determining thecytoplasmic chemical/physical environment of the cell. The use of highlysensitive EPR probe like LiNc-BuO, capable of measuring intracellularpO₂ with a greater sensitivity offers advantages in biological EPRoximetry. The data presented in this study demonstrated that this novelEPR probe can be successfully employed for direct and efficientmeasurement of intracellular oxygen concentration with a sensitivity of0.1 mmHg in all cell types. The cells internalized with LiNc-BuO can beused as an important tool to monitor oxidative cellular functions and tostudy the cellular responses under pathophysiological and toxicologicalconditions.

Summary and Conclusions:

The intracellular oxygen concentration in BLMVECs was measured byinternalizing oxygen sensitive microparticulate spin probe (LiNc-BuO)using electron paramagnetic resonance oximetry. The method utilized amicroparticulate spin probe (LiNc-BuO) with a high sensitivity foroxygen, enabling accurate measurement of intracellular pO₂ in BLMVECs.We also determined the extracellular oxygen concentration using anotheroxygen sensitive oximetry probe, TAM, simultaneously. The effect ofagents which can alter oxygen concentration such as menadione andpotassium cyanide on oxygen gradient was also studied. The measurementswere performed in a volume of 20 μL containing 4000 cells (2×105cells/mL) in a gas permeable Teflon tube at room air and at 7.5% oxygen.The intracellular oxygen concentration in BLMVECs measured at room airby this technique was 194 μM and extracellular oxygen concentration was220 μM with a gradient of 26 μM and an oxygen gradient of 16 μM was seenin cells exposed 7.5% oxygen. There was no significant difference inextra- and intracellular oxygen concentration during treatment withmenadione and potassium cyanide. In summary, we demonstrated that themeasurements of intracellular oxygen concentration and oxygen gradientcan be successfully performed using microparticulate-based EPR oximetrywith a fewer number of cells.

Crystal Structure of Li(BuO)₈Nc

The crystal structure of Li(BuO)₈Nc was studied by X-ray powderdiffraction (XRPD) analysis using a Bruker D8 diffractometer equippedwith a Cu Ka (λ=1.5406 Å) radiation tube, an incident beam Gemonochromator, and a Braun linear position sensitive detector (PSD). TheXRPD patterns were collected at room temperature varying the powdermounting conditions, angular step size, and counting time. Data werecollected using a conventional flat plate sample holder, azero-background silicon single crystal sample holder, and a spinningthin walled capillary. The various data sets were all fairly similar,suggesting that preferred orientation effects are not a significantproblem. In the capillary mode a noticeable background was observed over28 range 15-30° due to the amorphous nature of the glass capillary,whereas in the flat plate mode the peak intensities fell off rapidlywith (sin θ)/λ. Taking into account resolution and signal-to-noise, weelected to use a data set collected using a 1 mm diameter capillary fordetailed analysis. This scan covered the 28 range 2-−40° using a stepsize of 0.014347° and a counting time of 10 sec per step.

In order to determine the crystal symmetry and unit cell dimensions ofLi(BuO)₈Nc, the auto indexing software package CRYSFIRE [171] was used.Peak positions of first 24 reflections were fitted using the programXFIT [172] and exported to CRYSFIRE suite. Among the separatesubroutines included in CRYSFIRE, ITO12 [173] identified two triclinicstructures of very similar cell dimensions, with figures of merit (M),[174] 22 and 19. The space group was assumed to be the centric systemP−1 rather than P1, in light of the pronounced preference for the formerspace group among existing structures. Using the approximate cellparameters suggested by ITOI2, peak intensities were extracted by thewhole pattern fitting approach based on LeBail method [175] asimplemented in the GSAS software suite [176]. The LeBail fit gave a X2value of 5.16, an R_(wp) value of 0.0290, and refined cell parameters ofa=17.087(1) Å, b=18.792(1) Å, c=14.191(1) Å, a=113.577(6°,B=109.771(5)°, y=73.517(6)°, and volume=3871.5(5) Å³. The number offormula units per unit cell could be determined as Z=2 (LiC₈₀H₈₈N₈O₈,f.w. 1296.54, p=1.113 g/cm³) from packing considerations.

Following the autoindexing and whole pattern fitting stages, themolecular packing of Li(BuO)₈Nc was further studied by a globaloptimization approach implemented within DASH, [177] the details ofwhich are described elsewhere [178]. The peak intensities were extractedusing the Pawley method [179] implemented in DASH, although the latticeparameters were fixed at values determined in GSAS. Pawley refinementgave a profile X2 (Xpro2) value of 3.67 and a R_(wp) value of 0.0776.The LeBail and Pawley fits are shown in the supplementary information.Structure determination proceeded by means of the simulated annealingalgorithm provided in DASH using a starting model structure ofLi(BuO)₈Nc molecule constructed using the 3D Sketcher included inMaterial Studio [180]. The internal geometry of Li(BuO)₈Nc molecule didnot undergo further optimization, but the C—C or C—N bond lengths andthe size of naphthalocyanine ring were examined and found to be verysimilar to those in metal naphthalocyaninates of nickel, copper, or zinc[181]. Since the number of atoms in the asymmetric unit, 185, exceedsthe default values of DASH, which can handle up to 150 atoms in anasymmetric unit, the input structure was modified by removing allhydrogen atoms. The resulting molecular fragment consists of 96 atomsbut still retains 93% mass of the original Li(BuO)₈Nc molecule (see FIG.17).

Using the rigidity of the molecule as a chemical constraint, the crystalstructure of Li(BuO)₈Nc molecule can be described by 3 positionalvariables and 3 independent rotational parameters. In addition, each ofthe eight n-butoxy chains has 4 conformational degrees of freedom, for atotal of 32 unknown torsion angles. In a single simulated annealing runof DASH, 107 movements were made adjusting the above 38 variables togenerate a theoretical pattern that best matches the experimental data.The initial 45 trial runs, in which no constraints were imposed, did notlead to a straightforward solution possibly because the structure hastoo many flexible bonds, the combinations of which cannot be thoroughlyexamined. However a careful examination of output structures revealedtwo favored regions for the position (center of mass) of the molecule,as shown in the plot of positional parameters from 20 output resultswith lowest χpro² (see FIG. 18). Within both convergent groups, theorientations of naphthalocyanine rings differed only slightly amongtrials. The reproducible location and orientation of thenaphthalocyanine ring suggests that the crystal structure adopts one ofthese two molecular packing arrangements; referred to as type-I for thegroup centered at (0.05, 0.03, 0.37) and type-II for the group centeredat (0.44, 0.02, 0.15).

The results of the DASH structure solution attempts described in thepreceding paragraph indicate that given the complexity of the moleculein question and the limited resolution of the X-ray diffraction data itis not possible to obtain a complete structure solution. However, thereis a strong indication we can determine the approximate location andorientation of the napthalocyanine ring. To further investigate thisassumption we attempted structure solution using simpler modelstructures in which the —OBu groups are replaced by —OC_(n)H_(2n+l)(n=0, 1, 2, 3). Omission of all or part of the alkyl chains leaves 65%(—OH), 74% (—OMe), 83% (—OEt), and 91% (—OPr) of the total mass of theactual compound. While it is possible that this omission may mislead thestructure solution process, we felt that omission of atoms that cannotbe reliably located in the structure solution process may help to avoidfalling into false minima with regard to the location and orientation ofthe napthalocyanine ring. It was observed that reducing the total numberof conformational variables in this manner, leads to a significantimprovement in the reproducibility of the simulated annealing process.It is noteworthy that the solutions from all four cases agreed well bothin the position and orientation of the naphthalocyanine ring, with thetype-II solution. The χ_(pro) ² values gradually increased with thedecrease of the number of carbon atoms in the side chains: —OPr, ˜170,—OEt, ˜230, —OMe, ˜260, and —OH, ˜290. These results strongly suggestthat the type-II model describes the molecular packing of thenaphthalocyanine rings, and also that the alkoxy chains providenon-negligible contributions to the diffraction pattern.

In the subsequent step, we decided to fine-tune the stacking structureof Li(OBu)₈Nc by imposing constraints on the external degrees of freedomas obtained from above exploratory trials. The center of mass ofLi(OBu)₈Nc was confined to a volume defined by 0.35≦×≦0.5, 0≦y≦0.15,0.05≦z≦0.2, and the quarternians, Q_(i) (i=0, 1, 2, 3; −1≦Q_(i)≦1),[182] were limited to have a width of 0.4, while the thirty two torsionangles were allowed to vary freely. From 10 trials under the abovecontrolled condition, solutions were obtained with X_(pro) ² values of128.5˜147.2. The positions and orientations of the rigid ring part werealmost invariant according to the constraints, but the conformations ofeight butoxy chains were rather featureless. Consequently, a meaningfulpattern could not be ascertained. The various and irregularconformations of the butoxy chains must be responsible for the high anddispersed X_(pro) ² values. The refinement profile for the best-fitresult obtained with a X_(pro) ² value of 128.5, is given insupplementary information.

At this stage it is instructive to consider the quality of the data andthe solution, in a manner similar to that used in proteincrystallography. The useful information in the diffraction data beginsto die off at ˜26° 2θ (see Supplementary information), which correspondsto a d-spacing of 3.4 A. Consequently, it is futile to hope for astructure solution from this diffraction data that accurately reproducesthe crystal structure with atomic resolution. A more realisticexpectation would be to attempt to extract the molecular packing diagramof the planar naphthalocyanine rings. That would entail accuratelyfitting the diffraction pattern out to a d-spacing corresponding to theintermolecular spacing. In many metal naphthalocyaninates, the shortestinterplanar distances are ˜3.3 Å [181] but in the Li(OBu)₈Nc the butoxychains are likely to increase the intermolecular spacing. Our results(described in more detail below) suggest that the napthalocyaninemolecules are roughly 5 Å apart. Therefore, the information that we canhope to reliably extract from the diffraction pattern is contained inthe 2θ range 3.2-18.60 (d>4.8 Å). Building on this premise we proceededto analyze this low angle region of the diffraction pattern using thesimulated annealing algorithms in DASH. The solution obtained possessesa very low X_(pro) ² value of 24, and once again the type II model forthe location and orientation of the naphthalocyanine ring was obtained.The refinement profile for 3.2-18.60 range is shown in FIG. 19. Thisresult combined with the results using truncated alkoxy chains givecompelling proof that the approximate molecular packing diagramcorresponds to the type-II solution, while the type-I solutionrepresents a false minimum that results from the failure to accuratelydetermine the orientations of the n-butoxy chains. The high X_(pro) ²value that is obtained from analysis of the entire pattern can be tracedto the poor fit at higher 2θ region, where the coherence related to theorientation of the butoxy chains becomes increasingly important. Clearlythere is some degree of order in the orientation of these chains, butthe vast number of conformations that could be adopted prevents us fromextracting this information. To obtain this information single crystalstudies are probably necessary. Alternatively, energy minimizationalgorithms may be able to find the most favorable conformation given thedimensions of the unit cell and the approximate location of thenapthalocyanine ring.

FIG. 20 shows the obtained crystal structure of Li(OBu)₈Nc viewed alongthe three crystallographic axes. It can be noted that the molecules arearranged to form infinite channels along a- and c-axes. These channelsinterpenetrate each other at an angle of 109.8°, which is nearly thesame as the lattice angle, B. While the exact dimensions will besensitive to the conformation of the n-butoxy groups, thecross-sectional dimensions of the channels are not smaller than 8.1×9 Åand 4.6×5.7 Å along a- and c-axes, respectively. The presence of largeand interconnected voids in the crystal structure could allow facilediffusion of O₂ molecules, whose approximate size is 2.8×3.9 Å. Thisstructural motif is likely to be crucial to the sensor activity ofLi(OBu)₈Nc. The view along the b-axis (FIG. 20 b) shows that the planarrings of the Li(OBu)8Nc molecules are almost parallel to b-axis.Furthermore, it illustrates the columnar stacking of the molecules in a-and c-directions, which is responsible for the creation of channels.Within the columnar arrangements in both the directions, a dimer unit ofLi(OBu)₈Nc molecules is formed. The molecules in the dimer are slightlyglided away from the eclipsed conformation to have an interplanardistance of ˜4.8 Å and a Li—Li distance of 5.0 Å. Along the a-direction,the closest Li(OBu)₈Nc molecules from two neighboring dimers areseparated by an interplanar distance of 9.4 Å and a Li—Li distance of13.5 Å. On the other hand, in the c-direction, the planar spacingbetween the dimers is almost identical to the intra-dimer spacing of˜4.8 Å, and the adjacent dimers are glided from each other to have aLi—Li distance of 11.4 Å.

1. A particulate probe comprising a lithium phthalocyanine derivative ora radical thereof selected from the group consisting of:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)_(n)CH₃, O(CH₂)_(n)CH₂OH, O(CH₂)_(n)CH₂NH₂, O(CH₂)_(n)CH₂SH, andcombinations thereof; and, wherein n is from 1 to
 6. 2. The particulateprobe of claim 1, wherein the particulate probe has a size of up to 10microns.
 3. The particulate probe of claim 2, wherein the particulateprobe has a size of less than 0.22 microns.
 4. The particulate probe ofclaim 1, wherein the particulate probe has been derivatized to be amagnetic resonance imaging (MRI) probe, an electron spin resonance (ESR)probe, an electron paramagnetic resonance (EPR) probe, an electronparamagnetic resonance imaging (EPRI) probe, or a proton electron doubleresonance imaging (PEDRI) probe.
 5. The particulate probe of claim 1,wherein the probe comprises a compound of Formula 1 or a radicalthereof:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)_(n)CH₃, O(CH₂)_(n)CH₂OH, O(CH₂)_(n)CH₂NH₂, O(CH₂)_(n)CH₂ SH, andcombinations thereof; and, wherein n is from 1 to
 6. 6. The particulateprobe of claim 1, wherein the probe comprises a compound of Formula 2 ora radical thereof:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)nCH₃, O(CH₂)_(n)CH₂OH, O(CH₂)_(n)CH₂NH₂, O(CH₂)_(n)CH₂SH, andcombinations thereof; and, wherein n is from 1 to
 6. 7. The particulateprobe of claim 1, wherein the probe comprises a compound of Formula 5 ora radical thereof:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)_(n)CH₃, O(CH₂)_(n)CH₂0H, O(CH₂)_(n)CH₂NH₂, O(CH₂)_(n)CH₂SH, andcombinations thereof; and, wherein n is from 1 to
 6. 8. The particulateprobe of claim 1, wherein the probe comprises a compound of Formula 6 ora radical thereof:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)_(n)CH₃, O(CH₂)_(n)CH₂0H, O(CH₂)_(n)CH₂NH₂, O(CH₂)_(n)CH₂SH, andcombinations thereof; and, wherein n is from 1 to
 6. 9. A suspensioncomprising a particulate probe for MR imaging, the probe having oxygenactive centers, wherein the probe is a radical of a lithiumphthalocyanine derivative, and wherein the suspension is in a mediumselected from the group consisting of nonphysiological media,physiological media, buffers, and combinations thereof.
 10. Thesuspension of claim 9, wherein the particulate probe is selected fromthe group consisting of:

wherein R is selected from the group consisting of O(CH₂)_(n)CH₃,S(CH₂)_(n)CH₃, O(CH₂)_(n)CH₂OH, O(CH₂)_(n)CH₂NH₂, O(CH₂)_(n)CH₂SH, andcombinations thereof; and, wherein n is from 1 to
 6. 11. The suspensionof claim 9, further comprising a stabilizing agent, wherein thestabilizing agent is selected from the group consisting of amino acids,synthetic peptides, peptides of natural origin, proteins, sugars,carbohydrates, nucleic acid homopolymers, amino acid homopolymers, DNA,RNA, and combinations thereof; and wherein the stabilizing agent adheresto the radical probe without blocking the oxygen active centers.
 12. Thesuspension of claim 9, further comprising a stabilizing medium, whereinthe stabilizing medium is selected from the group consisting ofemulsions containing saturated fatty acids; emulsions containingunsaturated fatty acids; emulsions containing saturated and unsaturatedfatty acids; salts of emulsions containing saturated fatty acids; saltsof emulsions containing unsaturated fatty acids; salts of emulsionscontaining saturated and unsaturated fatty acids; diglycerides;triglycerides; bile salts; and combinations thereof.
 13. The suspensionof claim 9, further comprising a phospholipid, wherein the phospholipidencapsulates the radical probe without blocking the oxygen activecenters.
 14. The suspension of claim 13, wherein the phospholipid formsphospholipid liposomes which encapsulate the radical probe withoutblocking the oxygen active centers.
 15. The suspension of claim 14,wherein the phospholipid is selected from the group consisting ofcholesterol, phosphatidyl choline, phosphatidylethanolamine,phosphatidylserine, cardiolipin, and combinations thereof; and whereinthe phospholipid is in the form of unilamellar or multilamellarliposomes or vesicles.
 16. A method of measuring oxygen concentration,oxygen partial pressure, or oxygen metabolism in a specific tissue ororgan in a subject, the method comprising the steps of: (a)administering at least one particulate probe according to claim 1 to thesubject; and (b) applying a magnetic resonance (MR) spectroscopy orimaging technique for measuring O₂ concentration in tissues or organs ofthe subject.
 17. The method of claim 16, wherein the MR technique isselected from the group consisting of MRI, ESR, EPR, ERPI, and PEDRI.18. The method of claim 16, wherein the particulate probe or radicalthereof remains in the subject for at least 180 days.
 19. The method ofclaim 16, wherein the subject is a human subject.
 20. A method ofmeasuring nitric oxide (NO), separately or in combination with oxygen,in a specific tissue or organ of a subject, the method comprising thesteps of: (a) administering at least one particulate probe according toclaim 1 to the subject; and (b) applying a magnetic resonance (MR)spectroscopy technique for measuring NO concentration in tissues ororgans of the subject.